IL-12 Produced by Dendritic Cells Augments CD8 + Activation through the Production of the CCL1 and CCL17 This information is current as of September 23, 2021. Curtis J. Henry, David A. Ornelles, Latoya M. Mitchell, Kristina L. Brzoza-Lewis and Elizabeth M. Hiltbold J Immunol 2008; 181:8576-8584; ; doi: 10.4049/jimmunol.181.12.8576 http://www.jimmunol.org/content/181/12/8576 Downloaded from

References This article cites 75 articles, 35 of which you can access for free at:

http://www.jimmunol.org/content/181/12/8576.full#ref-list-1 http://www.jimmunol.org/

Why The JI? Submit online.

• Rapid Reviews! 30 days* from submission to initial decision

• No Triage! Every submission reviewed by practicing scientists

• Fast Publication! 4 weeks from acceptance to publication by guest on September 23, 2021

*average

Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts

The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2008 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

IL-12 Produced by Dendritic Cells Augments CD8؉ T Cell Activation through the Production of the Chemokines CCL1 and CCL171

Curtis J. Henry, David A. Ornelles, Latoya M. Mitchell, Kristina L. Brzoza-Lewis, and Elizabeth M. Hiltbold2

IL-12 family members are an important link between innate and adaptive immunity. IL-12 drives Th1 responses by augmenting IFN-␥ production, which is key for clearance of intracellular pathogens. IL-23 promotes the development of IL-17-producing CD4؉ T cells that participate in the control of extracellular pathogens and the induction of autoimmunity. However, recent studies have shown that these can modulate migration and cellular interactions. Therefore, we sought to determine ؉ the individual roles of IL-12 and IL-23 in naive CD8 T cell activation by addressing their ability to influence IFN-␥ production Downloaded from and cellular interaction dynamics during priming by Listeria monocytogenes-infected dendritic cells (DC). We found that IL-12 .was the major influencing the level of IFN-␥ production by CD8؉ T cells while IL-23 had little effect on this response In addition, we observed that IL-12 promoted longer duration conjugation events between CD8؉ T cells and DC. This enhanced cognate interaction time correlated with increased production of the chemokines CCL1 and CCL17 by WT but not IL-12-deficient DC. Neutralization of both chemokines resulted in reduced interaction time and IFN-␥ production, demonstrating their impor- ؉ ؉ tance in priming naive CD8 T cells. Our study demonstrates a novel mechanism through which IL-12 augments naive CD8 T http://www.jimmunol.org/ cell activation by facilitating production, thus promoting more stable cognate interactions during priming. The Journal of Immunology, 2008, 181: 8576–8584.

t is becoming increasingly clear that the initial signals CD8ϩ DC that promote long-term physical interactions leading to the T cells receive during priming impacts the subsequent pri- efficient priming of naive CD8ϩ T cells remains to be determined. mary and secondary responses generated in these cells (1– IL-12 is produced by mature DC in response to infection by I 3 13). Dendritic cells (DC) efficiently deliver the required signals to various intracellular pathogens (14, 22). Due to the potent effects ϩ activate naive CD8 T cells. Without DC, adaptive immune re- that IL-12 has on T cell activation, it is largely viewed as an im- by guest on September 23, 2021 sponses against several pathogens are not elicited (14–17). Their portant bridge between the innate and adaptive immune systems potent ability to stimulate naive T cells depends on their matura- (23–29). This cytokine influences several aspects of T cell activa- tion state (14, 17). Mature DC will present several activating li- tion, most notably IFN-␥ production, which plays a key role in the ϩ gands or secreted factors to naive CD8 T cells during priming resolution of intracellular pathogens such as Listeria monocyto- such as peptide-MHC complexes (signal 1), costimulatory mole- ϩ (Lm) (30, 31). Extensive research has been performed on cules (signal 2), and cytokines (signal 3) (18–20). CD8 T cells Lm and the immune responses required for resolving infection are encountering mature DC engage in long duration interactions, re- well documented (32). IL-12 is required to resolve high-dose in- sulting in the generation of highly functional effector cells (21). fection; however, in the absence of IL-12, increased IFN-␥ can However, an immature DC has a reduced capacity to prime naive ϩ compensate (30, 33, 34). Direct infection of DC with Lm in vitro CD8 T cells which correlates with shorter duration interactions results in production of p40, a cytokine subunit shared by IL-12 during activation (21). The full complement of factors delivered by and IL-23 (14). Furthermore, neutralization of p40 during priming of CD8ϩ T cells by Listeria-infected DC results in a significant decrease in the amount of IFN-␥ produced by these cells (14). Department of Microbiology and Immunology, Wake Forest University School of IFN-␥ plays multiple roles in resolving listerial infection including Medicine, Winston-Salem, NC 27157 activating and increasing target cell sensitivity to ly- Received for publication March 5, 2008. Accepted for publication October 13, 2008. sis by CD8ϩ T cells (8, 30, 35, 36). Thus, IL-12 production is a The costs of publication of this article were defrayed in part by the payment of page key hallmark of resolving Lm infection through its augmentation charges. This article must therefore be hereby marked advertisement in accordance of IFN-␥ production. with 18 U.S.C. Section 1734 solely to indicate this fact. The IL-12 family is composed of the cytokines IL-12, IL-23, and 1 This research was supported by the National Institutes of Health Research Project Grant Program (R01) Grant AI057770-01A1 (to E.M.H.). Additional support was IL-27 (reviewed in Refs. 24–29, 37–39). They are grouped together provided by the National Institutes of Health Predoctoral Fellowship Award for Mi- based on structural similarities between the cytokines and/or their nority Students (F31) Grant AI73245-01A1 (to C.J.H.). receptors. IL-12 and IL-23 share the p40 subunit. However, p35 is 2 Address correspondence and reprint requests to Dr. Elizabeth M. Hiltbold, Depart- ment of Microbiology and Immunology, 5109 Gray Building, Wake Forest University exclusively associated with IL-12 and a heterodimer of p19/p40 School of Medicine, Winston-Salem, NC 27157. E-mail address: bhiltbol@ makes up IL-23. The distinct biological functions of each cytokine wfubmc.edu in this family may be attributed to differences in the composition 3 Abbreviations used in this paper: DC, ; Lm, Listeria monocytogenes; of their receptors and distinct sets of downstream transcription WT, wild type; MFI, mean fluorescence intensity; ICS, intracellular cytokine staining. factors that are activated upon ligation. The main role attributed to Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00 IL-12 is the ability to induce and augment IFN-␥ production by www.jimmunol.org The Journal of Immunology 8577

ϩ CD4 T cells (driving a Th1-type response) as well as NK and i.v. (5 ϫ 105). At 18 h after infection, mice were sacrificed and inguinal, CD8ϩ T cells. IL-12 has been shown to increase CD8ϩ T cell lumbar, and mesenteric lymph nodes as well as spleens were harvested. RNA was isolated for chemokine analysis from DC following enrichment cytolysis, survival, proliferation, and influence the ability of these ϩ for CD11c cells. to migrate to inflammatory foci. A more recently rec- ognized activity of IL-23 is the ability to stimulate the production In vitro infection of DC ϩ of IL-17 by CD4 T cells. These Th17 cells are critical for the For T cell priming assays, DC were seeded at 2 ϫ 104/well and infected development of autoimmune diseases and immunity to certain ex- with WT Listeria (strain 10403S) at a multiplicity of infection (MOI) of 1. tracellular pathogens (24–29, 37–45). However, the role of IL-23 Four hours after infection, OVA peptide257–264 was added at a concentra- ␮ in the activation of naive CD8ϩ T cells is still being investigated. tion of 0.1 ng/ml (or as indicated) with chloramphenicol (10 g/ml) and gentamicin (10 ␮g/ml). Twenty-four hours after infection, OT-I T cells Since IL-12 and IL-23 have very potent effects on specific were added at 10:1 T:DC. For some experiments, OT-I T cells were stained immune responses, we wanted to determine the individual con- with CFSE before culture with DC. ϩ tributions of IL-12 and IL-23 on CD8 T cell priming by DC. Time-lapse video microscopy In addition, we wanted to identify the mechanism(s) through which these cytokines modulated naive CD8ϩ T cell activation. DC were prepared and infected as in T cell priming assays with the fol- lowing modifications. Day 6 WT, IL-12p35Ϫ/Ϫ, or IL-12p40Ϫ/Ϫ DC (106) To approach these goals, we used DC generated from mice Ϫ/Ϫ Ϫ/Ϫ were seeded in T25 flask and infected with Lm at a MOI of 1. After 4 h, lacking both IL-12 and IL-23 (p40 ) or only IL-12 (p35 ). antibiotics were added (chloramphenicol and gentamicin (10 ␮g/ml) and We found that IL-12, but not IL-23, augmented IFN-␥ produc- OVA peptide was added at a concentration of 0.1 ng/ml. At 24 h after tion by CD8ϩ T cells. We also found that IL-12 promoted infection, OT-I T cells were added to the flask at a ratio of 10:1 T cells:DC. ϩ longer duration interactions between CD8 T cells and Liste- For neutralization experiments, isotype control protein or neutralizing Abs Downloaded from to CCL1, CCL17, or IL-12/23 p40 were added to the flask at a concentra- ria-infected DC. To address the potential mechanisms through tion of 10 ␮g/ml. The total time of conjugation between individual CD8ϩ which IL-12 regulated T cell-DC interactions, we examined T cells and DC was determined by measuring the ligation (on) and detach- chemokine production in the presence or absence of IL-12. The ment (off) times of a T cell with DC conjugate. At least 50 interactions per production of CCL1 and CCL17 was increased by DC in the experiment were monitored. Interaction times are expressed as dissociation curves between T cell and DC with the percentage of T cells in conjugate presence of IL-12. Further studies determined that these che- vs total time of conjugation with DC (in min). A more gradual slope in- mokines directly increased the duration of conjugation as well dicates longer duration interactions between a population of T cells and http://www.jimmunol.org/ ϩ as IFN-␥ production by CD8 T cells. DC. Time-lapse phase-contrast images were recorded with an exposure time of 5-s frame intervals using an Olympus 1X70 enclosed camera with Materials and Methods an incubation chamber set at 37°C. Video recordings were captured over a 48-h period. Mice ELISAs C57BL/6, IL-12p35Ϫ/ϪBL/6-IL12atm/Jm, IL-12p40Ϫ/ϪBL/6-IL12btm/Jm, B6.129S1Il12rb2tm1Jm/J, OT-I, and OT-II TCR-transgenic mice specific for Culture supernatants from either DC infection/maturation assays or T cell- b b OVA257–264 presented by H-2K and OVA329–337 presented by I-A were priming assays were collected at the indicated times. These supernatants purchased from The Jackson Laboratory. P14 TCR-transgenic mice spe- were then probed for the presence of the following cytokines using ELISA

cific for the lymphocytic choriomeningitis virus gp peptide 33–41 were kits according to the manufacturer’s instructions: T cell activating 3 by guest on September 23, 2021 provided by Dr. J. M. Grayson (Wake Forest University School of Medi- (TCA3; CCL1) and and activation-regulated chemokine (CCL17) cine, Winston-Salem, NC). All mice were maintained and bred in the an- (DuoSet ELISA; R&D Systems), IL-12p40 (OptEIA kit; BD Biosciences), imal facility at Wake Forest University School of Medicine. IL-23 (eBioscience), and IFN-␥ (OptEIA kit; BD Biosciences). Cytokine/chemokine protein array Neutralizing Abs. To neutralize the activity of IL-12 and IL-23, anti-p40 WT, IL-12p35Ϫ/Ϫ, and IL-12p40Ϫ/Ϫ were infected with Lm at a MOI of 1. (clone C15.6; BioSource International) or just IL-23, anti-p19 (clone Twenty-four hours after infection, supernatants were harvested and probed G23-8; eBioscience) neutralizing Abs (10 ␮g/ml) were added before T cell for various cytokines and chemokines according to the manufacturer’s in- addition and maintained throughout the assay, unless otherwise indicated. structions (Raybiotech Mouse Cytokine Ab Array 3). Neutralizing Abs against the chemokines TARC/CCL17 (clone 110904; R&D Systems) and TCA-3/CCL1 (catalog no. AF845; R&D Systems) Real-time RT-PCR ␮ were added at 10 g/ml before T cell addition and maintained throughout Real-time PCR was performed according to the manufacture’s instructions the assay. (Applied Biosystems). Briefly, RNA was isolated from bone marrow-de- Surface and intracellular cytokine staining (ICS) Abs. Fluorescent Abs rived DC or DC enriched from the spleens and lymph nodes of Lm-infected measuring the expression of the mouse DC costimulatory molecules CD40 mice using the RNAqueous kit (Ambion). Once isolated, 20 ng of RNA of (clone 3/23), CD80 (clone 16-10A1), and CD86 (GL1) and the phenotypic each sample was used in each reaction. Primer and probe sets used to detect marker CD11c were purchased from BD Biosciences. IFN-␥ production by ϩ CCL1, CCL17, and ActB messages were purchased from Applied Biosys- CD8 OT-I T cells was measured using fluorescent Abs to CD8 (clone tems. TaqMan Universal PCR Master Mix was purchased from Applied 53-6.7; BD Pharmingen) and IFN-␥ APC (clone XMG1.2; BD Biosystems and nuclease-free water was purchased from Ambion. The re- Pharmingen). verse transcriptase used in these studies was Moloney murine leukemia DC propagation virus-reverse transcriptase, which was purchased from Invitrogen. The re- actions were performed and analyzed using the Applied Biosystems Prism Bone marrow-derived DC were generated as previously described (14). 7000 Sequence Detection System. Briefly, bone marrow was removed from the tibiae and femurs of 8- to Column enrichment of ex vivo DC 10-wk-old C57BL/6 (or strain indicated) mice. RBC were lysed and the progenitor cells (5 ϫ 105/ml) were resuspended and plated in RPMI 1640 DC were enriched (CDllcϩ selection) from the spleens and lymph nodes of containing 10% FCS supplemented with 10 ng/ml GM-CSF (generated Lm-infected mice according to the manufacturer’s instructions (Miltenyi from a recombinant baculovirus expression system). DC were cultured for Biotec). LS columns were used for the spleen and MS columns were used 6 days at 37°C in 5% CO2 and given fresh medium and cytokine on days for the lymph nodes. 2 and 4. DC used for the described experiments were between 90 and 95% CD11cϩ and expressed low levels of CD40, CD80, and CD86, which are Results characteristic of immature DC (data not shown). IL-12, but not IL-23 significantly augments IFN-␥ ϩ In vivo infection of mice production by CD8 T cells C57BL/6 (wild type (WT)) or IL-12-deficient (p35Ϫ/Ϫ) DC were mock Previous work from our laboratory and others has shown that ␥ ϩ treated (PBS only) or infected with 1 LD50 of Lm strain 10403S delivered IL-12 production increases IFN- production by CD8 T cells (3, 8578 ROLE OF CCL1 AND CCL17 IN CD8ϩ T CELL ACTIVATION

FIGURE 1. IL-12, but not IL-23 augments IFN-␥ production by naive CD8ϩ T cells. A, CFSE-stained OT-I T cells were primed by Listeria-infected OVA-pulsed WT, p35Ϫ/Ϫ,orp40Ϫ/Ϫ DC and IFN-␥ production was deter- mined by ICS on day 3 of priming. IFN-␥ MFI of T cells is denoted in the upper left corner. Contour plots are representative of 10 independent exper- iments. B, The combined data from eight independent flow cytometric ex- periments are graphed. The solid line indicates the amount of IFN-␥ T cells produced when primed in the absence of OVA peptide. The mean Ϯ SD is shown. C, IFN-␥ production by CD8ϩ T cells during priming measured by ELISA. CD8ϩ T cells were primed by either WT, p35Ϫ/Ϫ,orp40Ϫ/Ϫ DC and Downloaded from the amount of IFN-␥ that accumulated over 3 days was determined via ELISAs. The average concentration of IFN-␥ from three independent experi- ments is graphed. D, CFSE-stained OT-I T cells were primed by Listeria- http://www.jimmunol.org/ infected OVA-pulsed DC in the pres- ence of neutralizing Abs against IL-23 (anti-p19) or IL-12/23 (anti-p40) and IFN-␥ production was determined by ICS on day 3 of priming. Contour plots are representative of eight independent experiments. E, The combined data from 10 independent experiments are graphed. The solid line indicates the amount of IFN-␥ T cells produced by guest on September 23, 2021 when primed by WT DC in the absence of OVA peptide. The mean Ϯ SD is shown. Significance compared with the rat Ig or WT DC controls was deter- p Ͻ ,ء mined using Student’s t test with .p Ͻ 0.001 ,ءءء p Ͻ 0.01; and ,ءء ;0.05

14, 28, 29, 46–48). In addition, it has been reported that IL-12 IL-12 (p35Ϫ/Ϫ) alone (Fig. 1B). In addition, the amount of IFN-␥ augments the proliferative capacity of these cells (3, 18, 19). These secreted by CD8ϩ T cells over the 3-day priming period was re- conclusions were drawn primarily from neutralization studies in duced 3-fold compared with WT in the absence of IL-12 (Fig. 1C). which the p40 subunit of the cytokine was targeted; therefore, the Again, we observed no significant difference between the p35- and individual contributions of IL-12 or IL-23 to these responses could p40-deficient DC in their ability to influence IFN-␥ secretion over not be distinguished. To address the relative contributions of IL-12 this period. and IL-23 to CD8ϩ T cell activation, we primed naive CFSE- To more specifically address the role of IL-23, this cytokine was stained OT-I T cells with either WT, p35Ϫ/Ϫ (no IL-12), or p40Ϫ/Ϫ neutralized using an anti-p19 Ab. IL-23 neutralization did not signif- (no IL-12 or IL-23) DC. Before incubation with T cells, DC were icantly decrease OT-I IFN-␥ production on a per cell basis when infected with Listeria to induce cytokine production and were primed by WT DC (control IFN-␥ MFI, 262 and anti-p19 IFN-␥, MFI pulsed with OVA peptide. Infection of DC with Listeria resulted in 271; Fig. 1D). In contrast, when both IL-12 and IL-23 were neutral- low levels of IL-23 production from WT (53 pg/ml) and p35Ϫ/Ϫ ized (anti-p40), IFN-␥ production by OT-I was significantly de- DC (44 pg/ml), but not from p40Ϫ/Ϫ DC (7 pg/ml vs 10 pg/ml creased (control IFN-␥ MFI, 262 and anti-p40 IFN-␥ MFI, 74.6). produced by uninfected DC; data not shown). Neutralization of IL-23 alone or IL-12 and IL-23 in combination did On day 3 of priming, CD8ϩ T cell IFN-␥ production and pro- not alter proliferation (Fig. 1D). In addition, these results suggest that liferation were measured (Fig. 1A). Proliferation of CD8ϩ T cells IL-23 does not augment IFN-␥ production by CD8ϩ T cells and con- was not influenced by IL-12 or IL-23. However, in the absence of firm that IL-12 is a critical mediator of this process. IL-12, we observed a significantly decreased level of IFN-␥ pro- We next wanted to address the possibility that these observa- duced on a per cell basis (2-fold reduction in mean fluorescence tions could be attributed to changes in costimulatory molecule ex- intensity (MFI)). In the absence of both IL-12 and IL-23 (p40Ϫ/Ϫ), pression or Ag-presenting capacity of DC in the absence of IL-12 we observed the most pronounced decrease in the IFN-␥ MFI; yet, or IL-12/23. Therefore, we measured the up-regulation of it was not significantly less than that observed in the absence of costimulatory molecules (CD86, CD80, and CD40) and the Ag The Journal of Immunology 8579

FIGURE 2. IL-12 promotes long duration interactions with DC. A, OT-I were primed by Listeria-infected, OVA-pulsed DC for 48 h in vitro with control or anti-p40-neutralizing Abs. The duration of interaction between T cells and DC was determined via time-lapse video microscopy. B, OT-I were primed by WT, p35Ϫ/Ϫ,orp40Ϫ/Ϫ DC as described above and the duration of interaction between T cells and DC was determined via time-lapse video microscopy. Dissociation curves are combined data from at least three independent experiments per condition. The solid line in A and B represents 50% of the interactions analyzed. Significance compared with the WT DC ϩ OVA control samples was determined by Cox proportional hazard regression analysis. The neutralization of IL-12/23 (anti-p40) resulted in a significant decrease in the duration of interaction of OT-I (p Ͻ 0.01) with DC, which was further Downloaded from reduced in the absence of IL-12 (p35Ϫ/Ϫ DC; p Ͻ 0.001) or IL-12/23 (p40Ϫ/Ϫ DC; p Ͻ 0.001).

presentation capacity of all DC was determined by an OVA-spe- IL-12 augments CCL1 and CCL17 production by cific T cell hybridoma, B3Z, following listerial infection. We ob- Lm-infected DC http://www.jimmunol.org/ served similar levels of costimulatory molecule up-regulation and Ag IL-12 is not widely regarded as a chemotactic mediator, yet we Ϫ/Ϫ Ϫ/Ϫ presentation by Lm-infected WT, p35 , and p40 DC (data not observed enhanced cell-cell interaction when it was present. shown). Therefore, we decided to test whether IL-12 affected the expres- sion of chemokines by DC. To identify candidate chemokines pro- ϩ duced by DC in an IL-12-dependent manner, we used a cytokine/ IL-12 promotes long duration interactions between CD8 chemokine protein array. WT, p35Ϫ/Ϫ, and p40Ϫ/Ϫ DC were T cells and Lm-infected DC infected with Lm at a MOI of 1 and, 24 h after infection, the culture

Several studies have monitored the dynamics of interactions be- supernatants were subjected to this multiplex analysis. Interestingly, by guest on September 23, 2021 tween T cells and APCs to determine how these interactions im- expression of the two chemokines, CCL1 and CCL17, was markedly pact T cell proliferation and function (21, 49–51). What has be- reduced in the absence of IL-12 as well as IL-12/23 (data not shown). ϩ come clear is that full activation of naive CD8 T cell requires However, the lack of IL-12 and IL-23 did not appear to significantly long duration interactions with DC (21). Because IL-12 production alter the production of other chemokines as strongly, including peaks within 12 h of listerial infection and is known to influence MCP-1, MIP-1␣, MIP-1␥, or RANTES (data not shown). lymphocyte migration during inflammation (52, 53), we decided to We next wanted to more quantitatively measure the induction of test the effect of IL-12 on the duration of interactions between CCL1 and CCL17 by DC in the presence or absence of IL-12 or ϩ CD8 T cells and DC. DC were infected in a flask for 4 h, at which IL-23. As a first step, RNA was isolated from either WT, p35Ϫ/Ϫ, time OVA peptide and antibiotics were added. Twenty hours p40Ϫ/Ϫ, or DC lacking the IL-12R ␤2 chain (IL-12RϪ/Ϫ)at24h postinfection, anti-p40-neutralizing Abs and OT-I T cells were after infection with Lm. Real-time RT-PCR analysis was per- added to the culture. Time-lapse video microscopy was then per- formed to measure these chemokine messages. We found that the formed over a 48-h period and the on/off times of individual T CCL1 message was significantly reduced in the absence of IL-12, cell-DC interactions were measured. WT DC promoted long du- IL-12/23, and the IL-12R (all 4-fold reduced) compared with the ϩ ration interactions with CD8 T cells with 50% of the interac- levels found in WT DC (Fig. 3A). We also observed reduced tions lasting 127 min (Fig. 2A). Only transient interactions oc- CCL17 message in the absence of IL-12, IL-12/23, and the IL-12R curred in the absence of Ag. Neutralization of the p40 subunit (8-fold reduced) compared with the levels generated in Lm-in- of IL-12 and IL-23 significantly reduced the conjugation time fected WT DC (Fig. 3A). Taken together, these results indicate that ϩ between CD8 T cells and DC with 50% of the interactions IL-12 (but not IL-23) enhances the production of CCL1 and lasting only 27 min (Fig. 2A). CCL17 message by Lm-infected DC and that this enhancement is To determine the relative contributions of IL-12 and IL-23 to dependent on signaling through the IL-12 . this interaction, CD8ϩ T cells were primed by WT, p35Ϫ/Ϫ,or To more directly determine the role of IL-12 in augmenting p40Ϫ/Ϫ DC and the duration of conjugation was determined as CCL1 and CCL17 protein production, ELISA analysis was per- described above. In this study, we also observed that in the absence formed on the supernatants of WT or p35Ϫ/Ϫ DC infected with of IL-12 and IL-23 (p40Ϫ/Ϫ), the majority of interactions were of Listeria at 24 h after infection. We found that p35Ϫ/Ϫ DC pro- short duration, with 50% of the interactions lasting only 16 min duced significantly less CCL17 (3-fold reduction) at 24 h after (Fig. 2B). Again, there was no significant difference in the duration infection than WT DC (Fig. 3B). The amount of CCL1 produced of interaction in the absence of IL-12 (p35Ϫ/Ϫ) vs both IL-12 and by p35Ϫ/Ϫ DC was also reduced by 2-fold compared with WT DC IL-23 (p40Ϫ/Ϫ). This observation indicated that IL-12 could pro- (Fig. 3C); however, CCL1 secretion was very modest overall. mote long duration interactions between CD8ϩ T cells and DC and Additionally, we found significant reductions in secreted CCL17 that IL-23 did not significantly impact this interaction. (3-fold) and CCL1 (Ͼ2-fold) if DC lacked expression of the IL-12 8580 ROLE OF CCL1 AND CCL17 IN CD8ϩ T CELL ACTIVATION

T cells and DC in the presence of neutralizing Abs against CCL1 and CCL17. The neutralization of these chemokines resulted in a significant decrease in the duration of conjugation between the OT-I CD8ϩ T cells and DC compared with control samples (Fig. 4A). To determine whether this phenomenon could be generalized to other CD8ϩ T cells, not just OT-I, we included an additional source of naive CD8ϩ T cells, the P14 TCR-transgenic cells, spe-

cific for the lymphocytic choriomeningitis virus peptide gp33–41 presented by H-2Db. As in the case of the OT-I, we also observed a significant decrease in the duration of interaction of the P14 T cells with DC in the absence of CCL1 and CC17 (Fig. 4B). Thus, the diminished T cell-DC interaction time observed in the absence of IL-12 seems to correlate with similar decreases in interaction times observed when these two chemokines are neutralized. Neutralization of CCL1 and CCL17 significantly reduces the amount of IFN-␥ produced by CD8ϩ T cells Based on the observation that IL-12 augmented the production of

the chemokines CCL1 and CCL17 and that neutralization of these Downloaded from chemokines reduces T cell-DC interaction time, we decided to determine whether the neutralization of these chemokines also af- fected IFN-␥ production by CD8ϩ T cells. OT-I and P14 CD8ϩ T cells were primed by DC in the presence of the indicated neutral- izing Abs (Fig. 4, C and D). The neutralization of CCL1 or CCL17

alone resulted in only a modest (not statistically significant) re- http://www.jimmunol.org/ duction in the amount of IFN-␥ produced on a per cell basis by OT-I and P14 T cells. However, the neutralization of CCL1 and CCL17 in combination did significantly reduce IFN-␥ production by both T cell populations (2-fold less than the control). These observations indicate that CCL1 and CCL17 together augment IFN-␥ production by CD8ϩ T cells but that neither one alone sig- nificantly impacts this response. FIGURE 3. IL-12 increases the expression of CCL1 and CCL17 mes- Ϫ/Ϫ Ϫ/Ϫ sage and secretion in Lm-infected DC. WT, p35 , p40 , and IL- Neutralization of CCL1 and CCL17 does not reduce IFN-␥ by by guest on September 23, 2021 Ϫ Ϫ 12R / DC were mock treated or infected with Lm (MOI of 1), and RNA CD4ϩ T cells was isolated from these DC at 24 h after infection. A, CCL1 and CCL17 ␥ message levels were determined via real-time PCR. Data represent com- To determine whether CCL1 and CCL17 also augmented IFN- ϩ bined results from three independent experiments and the mean Ϯ SD is production by CD4 T cells, OVA-specific, OT-II-transgenic ϩ shown. B and C, WT, p35Ϫ/Ϫ, and IL-12RϪ/Ϫ DC were infected with Lm CD4 T cells were primed by Lm-infected DC in presence of at a MOI of 1. Chemokine secretion was measured 24 h later by ELISA. In neutralizing Abs against CCL1, CCL17, or both. Parallel experi- some experiments, rIL-12 was added back to IL-12-deficient DC (p35Ϫ/Ϫ) ments with OT-I T cells were used as positive controls. On day 3 to determine the effects this treatment had on CCL17 (B) and CCL1 pro- of priming, IFN-␥ production was measured by ELISA. We found duction (C). Significance was determined compared with the WT DC con- that neutralization of IL-12/23 (anti-p40) significantly reduced Ͻ ءءء Ͻ ءء Ͻ ء trol using Student’s t test with , p 0.05; , p 0.01; and , p IFN-␥ production by both OT-I and OT-II T cells (Fig. 5). IFN-␥ 0.001. ϩ production by CD8 T cells was also significantly reduced when CCL1 and CCL17 were neutralized in combination. However, the single or double neutralization of these chemokines did not sig- receptor (Fig. 3, B and C). Importantly, addition of rIL-12 to Lm- nificantly alter IFN-␥ production by CD4ϩ T cells. These results Ϫ Ϫ infected p35 / DC restored CCL1 and CCL17 production to levels indicate that unlike CD8ϩ T cells, CD4ϩ T cell production of comparable to WT DC (Fig. 3, B and C), further demonstrating that IFN-␥ is not augmented by CCL1 and CCL17. this is an IL-12-dependent enhancement. In contrast, the addition of rIL-23 to Lm-infected WT or IL-12-deficient DC did not alter CCL1 IL-12 augments CCL1 and CCL17 production from DC or CCL17 production (data not shown). Interestingly, the expression induced by listerial infection in vivo of the chemokine receptors for CCL1 (CCR8) or CCL17 (CCR4) by Having determined that CCL1 and CCL17 production was en- ϩ CD8 T cells remained unchanged whether IL-12 was added exog- hanced by IL-12 in vitro, we wanted to determine whether this enously or provided by DC (data not shown). These results reveal that enhancement was also observed in vivo. To address this question, IL-12 augments the production of CCL1 and CCL17 by Lm-infected WT or IL-12-deficient (p35Ϫ/Ϫ) mice were i.v. infected with Lm

DC at both the RNA and protein levels. (1 LD50). At 18 h after infection lymph nodes (pooled mesenteric, inguinal, and lumbar) were harvested. DC were enriched from Neutralization of CCL1 and CCL17 significantly reduces the ϩ Ͼ ϩ ϩ these organs using CD11c selection (cells were 85% CD11c ). duration of interaction between CD8 T cells and Once isolated, we purified RNA and determined the message lev- Lm-infected DC els for CCL1 and CCL17 using real-time PCR. To determine whether the chemokines CCL1 and CCL17 affect the We observed increased message levels for CCL1 and CCL17 in physical interaction between T cells and DC, time-lapse video mi- DC isolated from Lm-infected WT and p35Ϫ/Ϫ mice compared croscopy was used to measure the duration of interaction between with mock-treated mice (Fig. 6). As a normalization control, actin The Journal of Immunology 8581 Downloaded from http://www.jimmunol.org/

FIGURE 4. CCL1 and CCL17 augment T cell-DC interaction time and IFN-␥ production by CD8ϩ T cells. A and B, OT-I and P14 were primed by OVA or gp33–41-pulsed Listeria-infected DC for 72 h in vitro with control or anti-CCL1/CCL17-neutralizing Abs. The duration of interaction between T cells and DC was determined via time-lapse video microscopy. Dissociation curves are combined data from at least three independent experiments per condition. The solid line represents 50% of the interactions analyzed. Significance compared with the control samples was determined by Cox proportional hazard by guest on September 23, 2021 regression analysis. The neutralization of CCL1/17 resulted in a significant decrease in the duration of interaction of OT-I (p Ͻ 0.01) and P14 (p Ͻ 0.01) with DC compared with controls. C, OT-I and P14 T cells were primed by Listeria-infected, OVA, or gp33–41 peptide-pulsed DC for 72 h in vitro. Control, anti-CCL1, anti-CCL17, or a combination of anti-CCL1/17-neutralizing Abs was added when T cells were added with DC. D, The IFN-␥ MFI of T cells was determined at 72 h via ICS staining. Compiled data represent eight independent experiments for the OT-I T cells and four independent experiments for the P14 T cells. The relative statistical significance of either OT-I or P14 CD8ϩ T cell responses was determined compared with the same T cell in the .p Ͻ 0.001 ,ءءء p Ͻ 0.01; and ,ءء ;p Ͻ 0.05 ,ء absence of neutralization using Student’s t test with message levels were measured and were found to be equivalent in the message levels for CCL17 and a 4-fold decrease in CCL1 DC isolated from infected WT and p35Ϫ/Ϫ mice (data not shown). levels in DC isolated from the lymph nodes of IL-12-deficient In Lm-infected mice, we observed a greater than 6-fold decrease in mice compared with WT mice (Fig. 6). These data indicate that CCL1 and CCL17 production in response to listerial infection in

FIGURE 5. CCL1 and CCL17 do not augment IFN-␥ production by CD4ϩ T cells. OT-I and OT-II T cells were primed by Lm-infected DC in the presence or absence of anti-p40, anti-CCL1, anti-CCL17, or a combination of FIGURE 6. IL-12 augments CCL1 and CCL17 messages in DC during anti-CCL1/17-neutralizing Abs in vitro for 3 days. On day 3 of priming, su- Lm infection in vivo. WT and IL-12-deficient (p35Ϫ/Ϫ) DC were mock treated ␥ pernatants were filtered and assayed for the concentration of IFN- via (PBS) or infected with 1 LD50 of Lm i.v. At 18 h after infection, RNA was ELISAs. Histograms represent combined data from five independent experi- isolated from DC enriched from the lymph nodes of treated mice. Four mice ments for OT-I (A) and three independent experiments for OT-II T cells (B). per group were analyzed and data represent the mean Ϯ SD. CCL1 and Significance of neutralized samples was calculated in comparison to control CCL17 message levels were determined via real-time PCR. Significance was OT-I CD8ϩ T cells or OT-II CD4ϩ T cells without neutralization using Stu- assessed compared with DC isolated from WT mice using Student’s t test for .p Ͻ 0.001 ,ءءء p Ͻ 0.01; and ,ءء ;p Ͻ 0.05 ,ء p Ͻ 0.001. each gene with ,ءءء p Ͻ 0.01; and ,ءء ;p Ͻ 0.05 ,ء dent’s t test with 8582 ROLE OF CCL1 AND CCL17 IN CD8ϩ T CELL ACTIVATION vivo is enhanced by IL-12 to at least the same extent as that ob- CD4ϩ T cell proliferation and IFN-␥ production in vitro. Mice served in vitro. deficient in CCL17 production exhibit delayed allograft rejection and reduced hypersensitivity responses in vivo. Based on the lo- Discussion calization of CCL17- producing DC and their ability to promote The ability of IL-12 to promote strong Th1 responses by enhancing Th1 immune responses by CD4ϩ T cells, these DC are likely to be IFN-␥ production from various lymphocytes is well documented critical in the induction of anti-listerial CTL responses. Addition- (25, 27–29, 38). However, its ability to influence other parameters ally, patients with atopic were found to have signifi- of T cell activation, such as the physical interactions between im- cantly higher CCL1 levels when compared with the levels found in mune cells during priming, has not been well characterized. To our normal skin and other skin diseases (67). This chemokine, along knowledge, this is the first report which shows that IL-12 influ- with CXCL12, induced migration of T cells and DC, providing a ϩ ences the interactions between CD8 T cells and DC during prim- potential mechanism that would be important in linking innate and ing and points to a mechanism through which this effect is medi- adaptive immune responses, perhaps resulting in disease progres- ated. In our study, we found that IL-12 produced by DC sion (67). Thus, our observation that these chemokines enhance augmented the production of the chemokines CCL1 and CCL17. IFN-␥ production by CD8ϩ T cells adds to the growing body of These chemokines were found to promote longer duration inter- literature demonstrating the importance of these chemokines in the ϩ actions between CD8 T cells and Listeria-infected DC. These development of T cell responses. longer duration interactions correlated with increased IFN-␥ pro- ϩ Interestingly, while CCL1 and CCL17 were shown to regulate duction by CD8 T cells. Finally, neutralization of CCL1 and the level of IFN-␥ produced by CD8ϩ T cells, we observed little CCL17 resulted in a significant decrease in the production of effect of these chemokines on this response in CD4ϩ T cells. This Downloaded from ␥ IFN- and in the duration of cognate interaction. Taken together, difference may be due to distinct repertoires of chemokine recep- our findings highlight a novel mechanism in which IL-12 enhances tors expressed by the two T cell types or to differential require- chemokine production, prolonging T cell-DC interactions, and in- ments for the duration of cognate interaction. These questions are ␥ creasing the production of IFN- by T cells. the focus of ongoing study. It has been shown that IL-12 can influence T cell migration, ␥ ϩ ϩ Even though IL-12 augmented IFN- production through the based on observations that IL-12-conditioned CD8 and CD4 T mechanisms described above, we found that it did not play a sig- http://www.jimmunol.org/ cells are more responsive to chemokine stimulation (52). There- nificant role in increasing T cell proliferation. Thus, we have con- fore, an alternative explanation to our observed findings was that ϩ cluded that transient interaction between CD8 T cells and Ag- IL-12 influenced the expression of adhesion molecules or chemo- bearing DC are sufficient to induce T cell proliferation, which is in kine receptors expressed on CD8ϩ T cells. To address this possi- agreement with published literature (21). One potential explana- bility, we primed T cells by Listeria-infected WT, p35Ϫ/Ϫ,or tion why IL-12 does not play a significant role in augmenting T p40Ϫ/Ϫ DC for a period of 3 days. The surface expression of CCR8 cell proliferation when these lymphocytes are primed only by DC and CCR4 (the receptors for the chemokines CCL1 and CCL17, could be due to DC potency as APCs (14, 15, 17, 68–70). Listeria- respectively) as well as CCR5 and CXCR4, two chemokines

infected DC express high levels of costimulatory molecules such by guest on September 23, 2021 known to be important for the development of T cell responses (52, as CD80 and CD86 which augments CD8ϩ T cells proliferation 54–59), were monitored. We found no difference in the expression and cytokine production (14, 17). In addition, Lm-infected DC of these molecules over the 3-day priming period, regardless of the secrete cytokines such as IFN-␤ that can also be a potent signal 3 presence of IL-12. Additionally, expression of CD11a, CD69, and to T cells (14, 18–20, 71). Therefore, we feel that the effect of CD44, known mediators of adhesion during T cell activation (3, ϩ 60–65), were determined on CD8ϩ T cells on days 1, 2, and 3 of IL-12 on CD8 T cell proliferation changes based on other factors priming by flow cytometry. The expression levels of these mole- present during priming (the level of costimulation, other cytokines, cules were also not different in the presence or absence of IL-12 or and chemokines) that could play redundant roles with IL-12 in IL-23 (data not shown). In addition, we analyzed whether IL-12 augmenting this process (72–75). increased the surface expression of ICAM-1 on Listeria-infected Because IL-12 shares the p40 subunit with IL-23, we hypothe- DC. WT, p35Ϫ/Ϫ,orp40Ϫ/Ϫ DC were infected with Lm and, on sized that IL-23 could potentially augment the duration of inter- days 1, 2, and 3 after infection, the surface expression of ICAM-1 action between T cells and DC, correlating with the increased ␥ was measured via flow cytometry. The expression of ICAM-1 was IFN- production we observed. In our study, we found that IL-23 produced by DC did not augment IFN-␥ production by CD8ϩ T not influenced by IL-12 or IL-23 (data not shown). ϩ It has been determined that long duration interactions between cells or influence the interactions between CD8 T cells and DC CD8ϩ T cells and DC precedes effective activation of these cells during priming. From these observations, we conclude that IL-12 ϩ in vivo (21). The majority of these extended interactions occur has a greater impact on naive CD8 T cell activation than does within the initial 48 h of encounter (21). This observation corre- IL-23. However, since IL-23 activates multiple immune cells, its in lates with our results, which illustrate that the expression of the vivo role in the control of infection by intracellular bacterial patho- IL-12 receptor by CD8ϩ T cells peaks within the first 2 days of gens and T cell activation warrants further investigation. priming and is down-regulated after this period (data not shown). The observation that CCL17 is produced by DC in response to These observations may explain why CD8ϩ T cells are more sen- Lm infection in vivo was demonstrated several years ago (66). Our sitive to IL-12 within the first 2 days of activation when T cells are in vivo data confirm this finding and extend our understanding of engaged in long duration interaction with DC. the mechanistic basis of this response by demonstrating that this The roles of CCL1 and CCL17 in immune responses are only chemokine is produced by DC in an IL-12-dependent manner in beginning to be delineated, yet several recent reports indicate that response to listerial infection (Fig. 6). This result suggests that ϩ these chemokines play important roles in T cell activation. Fol- following in vivo infection with Listeria, CD8 T cells are likely lowing in vivo infection with Lm, Alferink et al. (66) observed an to remain in long-term cognate interaction with Ag-bearing DC accumulation of CCL17-producing DC in the lymph nodes and due to IL-12 and CCL1 and CCL17 production, enhancing their nonlymphoid organs of infected mice. In addition, bone marrow- expression of IFN-␥ and improving the ability of the host to clear derived DC producing CCL17 were better stimulators of naive the infection. The Journal of Immunology 8583

Our study has demonstrated a previously unrecognized mecha- 20. Curtsinger, J. M., J. O. Valenzuela, P. Agarwal, D. Lins, and M. F. Mescher. nism through which IL-12 regulates CD8ϩ T cell activation. Our 2005. Type I IFNs provide a third signal to CD8 T cells to stimulate clonal expansion and differentiation. J. Immunol. 174: 4465–4469. data, along with previously published studies, support the hypoth- 21. Hugues, S., L. Fetler, L. Bonifaz, J. Helft, F. Amblard, and S. Amigorena. 2004. esis that the delivery of IL-12 by DC to CD8ϩ T cells occurs most Distinct T cell dynamics in lymph nodes during the induction of tolerance and efficiently when these immune cells are in close proximity and immunity. Nat. Immunol. 5: 1235–1242. ϩ 22. Winzler, C., P. Rovere, M. Rescigno, F. Granucci, G. Penna, L. Adorini, when a large percentage of CD8 T cells express the receptor for V. S. Zimmermann, J. Davoust, and P. Ricciardi-Castagnoli. 1997. Maturation this cytokine. These observations further support our model in stages of mouse dendritic cells in -dependent long-term cultures. J. Exp. Med. which IL-12 (through the increased production of CCL1 and 185: 317–328. ϩ 23. Grohmann, U., R. Bianchi, M. L. Belladonna, C. Vacca, S. Silla, E. Ayroldi, CCL17) increases the physical interactions between CD8 T cells M. C. Fioretti, and P. Puccetti. 1999. IL-12 acts selectively on CD8␣-dendritic and DC when the T cells are most responsive to this cytokine. cells to enhance presentation of a tumor peptide in vivo. J. Immunol. 163: 3100–3105. However, it remains to be determined whether IL-12 and IL-23 24. Del Vecchio, M., E. Bajetta, S. Canova, M. T. Lotze, A. Wesa, G. Parmiani, and produced by DC affect the generation of potent memory or sec- A. Anichini. 2007. -12: biological properties and clinical application. ondary effector CD8ϩ T cell responses in the context of a bacterial Clin. Cancer Res. 13: 4677–4685. 25. Hunter, C. A. 2005. New IL-12-family members: IL-23 and IL-27, cytokines with infection. In addition, it will be important to determine whether divergent functions. Nat. Rev. Immunol. 5: 521–531. ϩ CCL1 and CCL17 govern naive CD8 T cell activation and gen- 26. Langrish, C. L., B. S. McKenzie, N. J. Wilson, R. de Waal Malefyt, eration of memory CTL in vivo, which could have major impli- R. A. Kastelein, and D. J. Cua. 2004. IL-12 and IL-23: master regulators of innate and adaptive immunity. Immunol. Rev. 202: 96–105. cations for vaccine design. 27. Watford, W. T., M. Moriguchi, A. Morinobu, and J. J. O’Shea. 2003. The biology of IL-12: coordinating innate and adaptive immune responses. Cytokine Growth

Factor Rev. 14: 361–368. Downloaded from Disclosures 28. Trinchieri, G. 2003. Interleukin-12 and the regulation of innate resistance and The authors have no financial conflict of interest. adaptive immunity. Nat. Rev. Immunol. 3: 133–146. 29. Trinchieri, G., S. Pflanz, and R. A. Kastelein. 2003. The IL-12 family of het- erodimeric cytokines: new players in the regulation of T cell responses. Immunity References 19: 641–644. 1. Kang, S. S., and P. M. Allen. 2005. Priming in the presence of IL-10 results in 30. Tripp, C. S., M. K. Gately, J. Hakimi, P. Ling, and E. R. Unanue. 1994. Neu- ϩ direct enhancement of CD8 T cell primary responses and inhibition of second- tralization of IL-12 decreases resistance to Listeria in SCID and C.B-17 mice: ary responses. J. Immunol. 174: 5382–5389. reversal by IFN-␥. J. Immunol. 152: 1883–1887. 2. Sallusto, F., and A. Lanzavecchia. 2002. The instructive role of dendritic cells on 31. Tripp, C. S., S. F. Wolf, and E. R. Unanue. 1993. and tumor http://www.jimmunol.org/ T-cell responses. Res. 4(Suppl. 3):S127–S132. necrosis factor ␣ are costimulators of gamma production by natural 3. Chang, J., J. H. Cho, S. W. Lee, S. Y. Choi, S. J. Ha, and Y. C. Sung. 2004. IL-12 killer cells in severe combined immunodeficiency mice with listeriosis, and in- priming during in vitro antigenic stimulation changes properties of CD8 T cells terleukin 10 is a physiologic antagonist. Proc. Natl. Acad. Sci. USA 90: and increases generation of effector and memory cells. J. Immunol. 172: 3725–3729. 2818–2826. 32. Pamer, E. G. 2004. Immune responses to Listeria monocytogenes. Nat. Rev. Im- 4. Chang, J., S. Y. Choi, H. T. Jin, Y. C. Sung, and T. J. Braciale. 2004. Improved munol. 4: 812–823. effector activity and memory CD8 T cell development by IL-2 expression during 33. Buchmeier, N. A., and R. D. Schreiber. 1985. Requirement of endogenous in- experimental respiratory syncytial virus infection. J. Immunol. 172: 503–508. terferon-␥ production for resolution of Listeria monocytogenes infection. Proc. 5. Langenkamp, A., M. Messi, A. Lanzavecchia, and F. Sallusto. 2000. Kinetics of Natl. Acad. Sci. USA 82: 7404–7408. dendritic cell activation: impact on priming of TH1, TH2, and nonpolarized T 34. Portnoy, D. A., R. D. Schreiber, P. Connelly, and L. G. Tilney. 1989. Gamma cells. Nat. Immunol. 1: 311–316. interferon limits access of Listeria monocytogenes to the cytoplasm. by guest on September 23, 2021 6. Joshi, N. S., and S. M. Kaech. 2008. Effector CD8 T cell development: a bal- J. Exp. Med. 170: 2141–2146. ancing act between memory cell potential and terminal differentiation. J. Immu- 35. Tripp, C. S., and E. R. Unanue. 1995. Macrophage production of IL12 is a critical nol. 180: 1309–1315. ϩ link between the innate and specific immune responses to Listeria. Res. Immunol. 7. Harty, J. T., and V. P. Badovinac. 2008. Shaping and reshaping CD8 T-cell 146: 515–520. memory. Nat. Rev. Immunol. 8: 107–119. 36. Harty, J. T., L. L. Lenz, and M. J. Bevan. 1996. Primary and secondary immune 8. Messingham, K. A., V. P. Badovinac, A. Jabbari, and J. T. Harty. 2007. A role responses to Listeria monocytogenes. Curr. Opin. Immunol. 8: 526–530. for IFN-␥ from antigen-specific CD8ϩ T cells in protective immunity to Listeria 37. Cordoba-Rodriguez, R., and D. M. Frucht. 2003. IL-23 and IL-27: new members monocytogenes. J. Immunol. 179: 2457–2466. of the growing family of IL-12-related cytokines with important implications for 9. Joshi, N. S., W. Cui, A. Chandele, H. K. Lee, D. R. Urso, J. Hagman, L. Gapin, therapeutics. Expert Opin. Biol. Ther. 3: 715–723. and S. M. Kaech. 2007. Inflammation directs memory precursor and short-lived ϩ 38. Goriely, S., and M. Goldman. 2007. The interleukin-12 family: new players in effector CD8 T cell fates via the graded expression of T-bet transcription factor. transplantation immunity? Am. J. Transplant. 7: 278–284. Immunity 27: 281–295. ϩ 39. Sun, J., M. Walsh, A. V. Villarino, L. Cervi, C. A. Hunter, Y. Choi, and 10. Badovinac, V. P., and J. T. Harty. 2007. Manipulating the rate of memory CD8 E. J. Pearce. 2005. TLR ligands can activate dendritic cells to provide a MyD88- T cell generation after acute infection. J. Immunol. 179: 53–63. dependent negative signal for Th2 cell development. J. Immunol. 174: 742–751. 11. Wong, P., M. Lara-Tejero, A. Ploss, I. Leiner, and E. G. Pamer. 2004. Rapid development of T cell memory. J. Immunol. 172: 7239–7245. 40. Stockinger, B., M. Veldhoen, and B. Martin. 2007. Th17 T cells: Linking innate 12. Busch, D. H., K. M. Kerksiek, and E. G. Pamer. 2000. Differing roles of inflam- and adaptive immunity. Semin. Immunol. 19: 353–361. mation and antigen in T cell proliferation and memory generation. J. Immunol. 41. Weaver, C. T., R. D. Hatton, P. R. Mangan, and L. E. Harrington. 2007. IL-17 164: 4063–4070. family cytokines and the expanding diversity of effector T cell lineages. Annu. 13. Lauvau, G., S. Vijh, P. Kong, T. Horng, K. Kerksiek, N. Serbina, R. A. Tuma, Rev. Immunol. 25: 821–852. and E. G. Pamer. 2001. Priming of memory but not effector CD8 T cells by a 42. Weaver, C. T., L. E. Harrington, P. R. Mangan, M. Gavrieli, and K. M. Murphy. killed bacterial vaccine. Science 294: 1735–1739. 2006. Th17: an effector CD4 T cell lineage with ties. Immunity 14. Brzoza, K. L., A. B. Rockel, and E. M. Hiltbold. 2004. Cytoplasmic entry of 24: 677–688. Listeria monocytogenes enhances dendritic cell maturation and T cell differen- 43. Mangan, P. R., L. E. Harrington, D. B. O’Quinn, W. S. Helms, D. C. Bullard, tiation and function. J. Immunol. 173: 2641–2651. C. O. Elson, R. D. Hatton, S. M. Wahl, T. R. Schoeb, and C. T. Weaver. 2006. ␤ 15. Jung, S., D. Unutmaz, P. Wong, G. Sano, K. De los Santos, T. Sparwasser, S. Wu, Transforming growth factor- induces development of the TH17 lineage. Nature S. Vuthoori, K. Ko, F. Zavala, E. G. Pamer, D. R. Littman, and R. A. Lang. 2002. 441: 231–234. In vivo depletion of CD11cϩ dendritic cells abrogates priming of CD8ϩ T cells 44. Harrington, L. E., P. R. Mangan, and C. T. Weaver. 2006. Expanding the effector by exogenous cell-associated antigens. Immunity 17: 211–220. CD4 T-cell repertoire: the Th17 lineage. Curr. Opin. Immunol. 18: 349–356. 16. Belz, G. T., K. Shortman, M. J. Bevan, and W. R. Heath. 2005. CD8␣ϩ dendritic 45. Harrington, L. E., R. D. Hatton, P. R. Mangan, H. Turner, T. L. Murphy, ϩ cells selectively present MHC class I-restricted noncytolytic viral and intracel- K. M. Murphy, and C. T. Weaver. 2005. -producing CD4 effector lular bacterial antigens in vivo. J. Immunol. 175: 196–200. T cells develop via a lineage distinct from the T helper type 1 and 2 lineages. Nat. 17. Muraille, E., R. Giannino, P. Guirnalda, I. Leiner, S. Jung, E. G. Pamer, and Immunol. 6: 1123–1132. ϩ G. Lauvau. 2005. Distinct in vivo dendritic cell activation by live versus killed 46. Berg, R. E., C. J. Cordes, and J. Forman. 2002. Contribution of CD8 T cells to Listeria monocytogenes. Eur. J. Immunol. 35: 1463–1471. innate immunity: IFN-␥ secretion induced by IL-12 and IL-18. Eur. J. Immunol. 18. Curtsinger, J. M., D. C. Lins, and M. F. Mescher. 2003. Signal 3 determines 32: 2807–2816. tolerance versus full activation of naive CD8 T cells: dissociating proliferation 47. Berg, R. E., E. Crossley, S. Murray, and J. Forman. 2005. Relative contributions and development of effector function. J. Exp. Med. 197: 1141–1151. of NK and CD8 T cells to IFN-␥ mediated innate immune protection against 19. Curtsinger, J. M., C. S. Schmidt, A. Mondino, D. C. Lins, R. M. Kedl, Listeria monocytogenes. J. Immunol. 175: 1751–1757. M. K. Jenkins, and M. F. Mescher. 1999. Inflammatory cytokines provide a third 48. Fantuzzi, G., D. A. Reed, and C. A. Dinarello. 1999. IL-12-induced IFN-␥ is signal for activation of naive CD4ϩ and CD8ϩ T cells. J. Immunol. 162: dependent on caspase-1 processing of the IL-18 precursor. J. Clin. Invest. 104: 3256–3262. 761–767. 8584 ROLE OF CCL1 AND CCL17 IN CD8ϩ T CELL ACTIVATION

49. Shakhar, G., R. L. Lindquist, D. Skokos, D. Dudziak, J. H. Huang, 62. Slifka, M. K., and J. L. Whitton. 2000. Activated and memory CD8ϩ T cells can M. C. Nussenzweig, and M. L. Dustin. 2005. Stable T cell-dendritic cell inter- be distinguished by their cytokine profiles and phenotypic markers. J. Immunol. actions precede the development of both tolerance and immunity in vivo. Nat. 164: 208–216. Immunol. 6: 707–714. 63. Geijtenbeek, T. B., R. Torensma, S. J. van Vliet, G. C. van Duijnhoven, 50. Hurez, V., A. Saparov, A. Tousson, M. J. Fuller, T. Kubo, J. Oliver, G. J. Adema, Y. van Kooyk, and C. G. Figdor. 2000. Identification of DC-SIGN, B. T. Weaver, and C. T. Weaver. 2003. Restricted clonal expression of IL-2 by a novel dendritic cell-specific ICAM-3 receptor that supports primary immune naive T cells reflects differential dynamic interactions with dendritic cells. J. Exp. responses. Cell 100: 575–585. Med. 198: 123–132. 64. Krummel, M., C. Wulfing, C. Sumen, and M. M. Davis. 2000. Thirty-six views 51. Jabbari, A., K. L. Legge, and J. T. Harty. 2006. T cell conditioning explains early of T-cell recognition. Philos. Trans. R. Soc. Lond. B. Biol. Sci. 355: 1071–1076. disappearance of the memory CD8 T cell response to infection. J. Immunol. 177: 65. Feng, H., D. Zhang, D. Palliser, P. Zhu, S. Cai, A. Schlesinger, L. Maliszewski, 3012–3018. and J. Lieberman. 2005. Listeria-infected myeloid dendritic cells produce IFN-␤, 52. Iwasaki, M., T. Mukai, P. Gao, W. R. Park, C. Nakajima, M. Tomura, priming T cell activation. J. Immunol. 175: 421–432. H. Fujiwara, and T. Hamaoka. 2001. A critical role for IL-12 in CCR5 induction on T cell receptor-triggered mouse CD4ϩ and CD8ϩ T cells. Eur. J. Immunol. 31: 66. Alferink, J., I. Lieberam, W. Reindl, A. Behrens, S. Weiss, N. Huser, K. Gerauer, 2411–2420. R. Ross, A. B. Reske-Kunz, P. Ahmad-Nejad, H. Wagner, and I. Forster. 2003. 53. Aliberti, J., C. Reis e Sousa, M. Schito, S. Hieny, T. Wells, G. B. Huffnagle, and Compartmentalized production of CCL17 in vivo: strong inducibility in periph- A. Sher. 2000. CCR5 provides a signal for microbial induced production of IL-12 eral dendritic cells contrasts selective absence from the spleen. J. Exp. Med. 197: by CD8␣ϩ dendritic cells. Nat. Immunol. 1: 83–87. 585–599. 54. Nguyen, D. H., B. Giri, G. Collins, and D. D. Taub. 2005. Dynamic reorgani- 67. Gombert, M., M. C. Dieu-Nosjean, F. Winterberg, E. Bunemann, R. C. Kubitza, zation of chemokine receptors, cholesterol, lipid rafts, and adhesion molecules to L. Da Cunha, A. Haahtela, S. Lehtimaki, A. Muller, J. Rieker, et al. 2005. CCL1- sites of CD4 engagement. Exp. Cell Res. 304: 559–569. CCR8 interactions: an axis mediating the recruitment of T cells and Langerhans- 55. Bromley, S. K., D. A. Peterson, M. D. Gunn, and M. L. Dustin. 2000. Cutting type dendritic cells to sites of atopic skin inflammation. J. Immunol. 174: edge: hierarchy of and TCR signals regulating T cell migra- 5082–5091. tion and proliferation. J. Immunol. 165: 15–19. 68. Badovinac, V. P., K. A. Messingham, A. Jabbari, J. S. Haring, and J. T. Harty. ϩ 56. Langenkamp, A., K. Nagata, K. Murphy, L. Wu, A. Lanzavecchia, and 2005. Accelerated CD8 T-cell memory and prime-boost response after dendrit- F. Sallusto. 2003. Kinetics and expression patterns of chemokine receptors in ic-cell vaccination. Nat. Med. 11: 748–756. Downloaded from ϩ human CD4 T lymphocytes primed by myeloid or plasmacytoid dendritic cells. 69. Zammit, D. J., L. S. Cauley, Q. M. Pham, and L. Lefrancois. 2005. Dendritic cells Eur. J. Immunol. 33: 474–482. maximize the memory CD8 T cell response to infection. Immunity 22: 561–570. 57. Sallusto, F., E. Kremmer, B. Palermo, A. Hoy, P. Ponath, S. Qin, R. Forster, 70. Kapsenberg, M. L. 2003. Dendritic-cell control of pathogen-driven T-cell polar- M. Lipp, and A. Lanzavecchia. 1999. Switch in chemokine receptor expression ization. Nat. Rev. Immunol. 3: 984–993. upon TCR stimulation reveals novel homing potential for recently activated T 71. Hernandez, J., S. Aung, K. Marquardt, and L. A. Sherman. 2002. Uncoupling of cells. Eur. J. Immunol. 29: 2037–2045. proliferative potential and gain of effector function by CD8ϩ T cells responding 58. Kim, C. H., L. Rott, E. J. Kunkel, M. C. Genovese, D. P. Andrew, L. Wu, and to self-antigens. J. Exp. Med. 196: 323–333.

E. C. Butcher. 2001. Rules of chemokine receptor association with T cell polar- http://www.jimmunol.org/ 72. Wong, P., and E. G. Pamer. 2001. Cutting edge: antigen-independent CD8 T cell ization in vivo. J. Clin. Invest. 108: 1331–1339. proliferation. J. Immunol. 166: 5864–5868. 59. Sallusto, F., D. Lenig, C. R. Mackay, and A. Lanzavecchia. 1998. Flexible pro- grams of chemokine receptor expression on human polarized T helper 1 and 2 73. Molon, B., G. Gri, M. Bettella, C. Gomez-Mouton, A. Lanzavecchia, lymphocytes. J. Exp. Med. 187: 875–883. A. C. Martinez, S. Manes, and A. Viola. 2005. T cell costimulation by chemokine 60. Curtsinger, J. M., D. C. Lins, and M. F. Mescher. 1998. CD8ϩ memory T cells receptors. Nat. Immunol. 6: 465–471. (CD44high, Ly-6Cϩ) are more sensitive than naive cells to (CD44low, Ly-6CϪ)to 74. Lanzavecchia, A., and F. Sallusto. 2001. Antigen decoding by T lymphocytes: TCR/CD8 signaling in response to antigen. J. Immunol. 160: 3236–3243. from synapses to fate determination. Nat. Immunol. 2: 487–492. 61. van Gisbergen, K. P., L. C. Paessens, T. B. Geijtenbeek, and Y. van Kooyk. 2005. 75. Nembrini, C., B. Abel, M. Kopf, and B. J. Marsland. 2006. Strong TCR signaling, Molecular mechanisms that set the stage for DC-T cell engagement. Immunol. TLR ligands, and cytokine redundancies ensure robust development of type 1 Lett. 97: 199–208. effector T cells. J. Immunol. 176: 7180–7188. by guest on September 23, 2021