T Cells + Memory CD8 Signaling in the Generation of Long-Lived A

A Novel Role of CD30/CD30 Ligand Signaling in the Generation of Long-Lived Memory CD8+ T Cells

This information is current as Hitoshi Nishimura, Toshiki Yajima, Hiromi Muta, Eckhard of September 24, 2021. R. Podack, Kenzaburo Tani and Yasunobu Yoshikai J Immunol 2005; 175:4627-4634; ; doi: 10.4049/jimmunol.175.7.4627 http://www.jimmunol.org/content/175/7/4627 Downloaded from

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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 © 2005 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

A Novel Role of CD30/CD30 Ligand Signaling in the Generation of Long-Lived Memory CD8؉ T Cells1

Hitoshi Nishimura,* Toshiki Yajima,* Hiromi Muta,† Eckhard R. Podack,‡ Kenzaburo Tani,† and Yasunobu Yoshikai2*

؉ ؉ Memory CD8 T cells can be divided into two subsets, central memory (TCM) and effector memory (TEM) CD8 T cells. We found that CD30, a member of the TNFR-associated factor (TRAF)-linked TNFR superfamily, signaling is involved in differentiation of ؉ ؉ long-lived CD8 TCM cells following Listeria monocytogenes infection. Although CD8 TEM cells transiently accumulated in the ؊/؊ ؉ nonlymphoid tissues of CD30 ligand (CD153 ) mice after infection, long-lived memory CD8 TCM cells were poorly generated in these mice. CCR7 mRNA expression was down-regulated in CD8؉ T cells of the spleen of CD153؊/؊ mice in vivo and the ؉ expression was up-regulated in CD8 TEM cells by anti-CD30 mAb cross-linking in vitro. These results suggest that CD30/CD30 ؉ ligand signaling plays an important role in the generation of long-lived memory CD8 T cells at least partly by triggering homing Downloaded from receptors for TCM cells. The Journal of Immunology, 2005, 175: 4627–4634.

eneration and maintenance of long-lived CD8ϩ T cell differentially during primary immune response depending on the memory are important goals of vaccination because they conditions of activation (5). However, very little is known about G can provide protection against reinfection of many signaling that regulates the differentiation pathway of these mem- pathogens, including intracellular bacteria (1). Immune response ory T cells. http://www.jimmunol.org/ against the pathogens depends on the ability of lymphocytes to Signaling via the TNFR superfamily regulates the fate of acti- migrate to appropriate places within the body to find their cognate vated T cells (6). Members of the TNFR superfamily such as Fas ϩ Ags (2). Memory CD8 T cells have been divided into two subsets and TNFR type I, which contain a death domain in the cytoplasmic based on their anatomical location, expression of cell surface tail, are responsible for activation-induced T cell death (7), markers, and effector functions (3). Memory T cells expressing whereas members having no death domain such as CD40 and 3 homing receptors such as CD62 ligand (CD62L) and CCR7, CD70 play a key role in effective T cell immunity (6). For exam- which allow efficient homing to lymph nodes (LNs), are termed ple, generation of memory CD8ϩ T cells displaying an enhanced central memory (TCM) cells, whereas memory T cells lacking these capacity for cell division and cytokine secretion requires CD40L by guest on September 24, 2021 LN-homing receptors, which are located in nonlymphoid tissues, expression by CD4ϩ T cells (8–11). CD30, a member of the are termed effector memory (T ) cells. T cells have been re- ϩ EM CM TNFR superfamily, is expressed by activated or memory CD8 T ported to produce few effector molecules but to have homeostatic cells but not by resting B or T cells (12–14). A CD30 ligand proliferative capacity in response to IL-15 (4, 5). In contrast, T EM (CD30L, CD153) is a 40-kDa type II membrane-associated gly- cells are thought to facilitate their entry into infected tissues ex- coprotein belonging to the TNF family (15). CD153 is expressed pressing cognate effector molecules for these receptors (4, 5). It is on macrophages, dendritic cells, and B cells (6, 14–16). Little is suggested that T and T cells do not necessarily represent CM EM known about the role of CD30/CD30L signaling in generation of distinct subsets and that they are part of a continuum in a linear memory CD8ϩ T cells, although there are several lines of evidence naive effector-TEM-TCM differentiation pathway (4). In contrast, there is some evidence that T and T cells might be generated showing that the signaling regulates peripheral T cell response CM EM through controlling T cell survival and down-regulation of cytolytic capacity (17–22). *Division of Host Defense, Center for Prevention of Infectious Disease, and †Division We previously reported that gene expression of CCR7 is up- of Molecular Genetics, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan; and ‡Department of Microbiology and Immunology, University of regulated by CD30 stimulation in the human YT lymphoma cell Miami, Miami, FL 33101 line (23, 24). This raises the possibility that CD30/CD30L signal- Received for publication August 10, 2004. Accepted for publication July 14, 2005. ing plays an important role in differentiation of memory T cell The costs of publication of this article were defrayed in part by the payment of page subsets following Ag exposure. In the present study, we found charges. This article must therefore be hereby marked advertisement in accordance with CD153Ϫ/Ϫ mice that CD30/CD30L signaling is involved in with 18 U.S.C. Section 1734 solely to indicate this fact. ϩ differentiation of CD8 TCM cells following exposure to a 1 This work was supported in part by a Grant-in Aid for Scientific Research on Priority Areas, Japan Society for the Promotion of Science, and by grants from the microbe. Japanese Ministry of Education, Science and Culture (to Y.Y.), Yakult Bioscience Foundation (to Y.Y.), Uehara Memorial Foundation (to Y.Y.), Nakamura Jishirou Foundation (to H.N.), Kurozumi Medical Foundation (to H.N.), and Kanzawa Med- ical Research Foundation (to H.N.). Materials and Methods 2 Address correspondence and reprint requests to Dr. Yasunobu Yoshikai, Division of Mice Host Defense, Center for Prevention of Infectious Disease, Medical Institute of Bio- regulation, Kyushu University, Fukuoka 812-8582, Japan. E-mail address: Age- and sex-matched BALB/c mice were obtained from Charles River [email protected] Breeding Laboratories. The generation and preliminary characterization of Ϫ/Ϫ 3 CD153 mice were previously described (24). All mice were maintained Abbreviations used in this paper: CD62L, CD62 ligand; LN, lymph node; TCM, central memory T cell; TEM, effector memory T cell; Tc, T cytotoxic cell; LLO, under specific pathogen-free conditions and were offered food and water ad listeriolysin O; CD30L, CD30 ligand; TRAF, TNFR-associated factor. libitum. All mice were used at 6–8 wk of age.

Ϫ ϩ ϩ Microorganism umns (Miltenyi Biotec). TEM (CD62L CD44 CD8 ) cells were nega- tively separated with anti-CD62L mAb microbeads and anti-CD4 mAb Listeria monocytogenes strain EGD was used in all experiments. Bacterial microbeads from nylon wool-enriched spleen T cells (Ͼ95% purity). Total virulence was maintained by serial passages in BALB/c mice. Mice were RNA was isolated from CD8ϩ T cells and from CD62LϪCD44ϩCD8ϩ T inoculated i.p. with various doses of viable L. monocytogenes in 0.2 ml of cells of CD153Ϫ/Ϫ mice and control mice with TRIzol reagent (Invitrogen PBS on day 0. The spleen and liver were removed from each mouse and Life Technologies). The first-strand cDNA synthesized from the total RNA separately placed on trypto-soya agar plates, and colonies were counted was amplified using 10 pmol of each primer specific for murine rRNA or after incubation for 24 h at 37°C. chemokine receptors. cDNA encoding chemokine receptors was analyzed by real-time PCR using a TaqMan PCR kit and an ABI PRISM 7000 Abs and reagents sequence detector thermal cycler according to the protocol recommended FITC-conjugated anti-CD44 (IM7), anti-CD69 (H1.2F3), and anti-IFN-␥ by the manufacturer (Applied Biosystems). (XMG1.2); PE-conjugated anti-CD8␣ (53-6.7), anti-CD44 (IM7), and anti- ␣ CD62L (MEL-14); and CyChrome-conjugated anti-CD8 (53-6.7) and anti- In vivo cytotoxicity assay CD4 (RM4-5) were purchased from BD Pharmingen. CyChrome and allophycocyanin-conjugated streptavidin were also obtained from BD Analysis of in vivo cytolytic activity was conducted basically according to Pharmingen. CFSE was purchased from Molecular Probes. the previously reported protocol (29, 30). BALB/c splenocytes were divided into two populations and labeled with either a high concentration (3 d Generation of H2-K tetramers ␮M) or a low concentration (0.3 ␮M) of CFSE. Next, CSFEhigh cells were pulsed with 10Ϫ6 M LLO for1hat37°C in the dark, whereas CFSElow MHC-peptide tetramers for staining of epitope-specific cells were gener- 91–99 cells remained nonpulsed. After washing, CFSEhigh cells were mixed with ated as previously described (25, 26). Briefly, purified H chain and ␤ - 2 equal numbers of CSFElow cells, and a suspension of 2 ϫ 107 of these cells microgrobulin were dissolved in 8 M urea and diluted in a refolding buffer was i.v. injected into each mouse. Spleens from recipients were obtained containing a high concentration of synthetic peptide listeriolysin O Downloaded from 15 h later for flow cytometric analysis to measure in vivo killing as indi- (LLO) (27) or JAK1 self-peptide (28) to generate monomeric soluble 91–99 cated by loss of the CFSEhigh Ag-pulsed population relative to the control H2-Kd-peptide complexes. Biotinylation and tetramerization of the het- CFSElow population. Percentage of specific lysis was calculated according erodimer were performed as described by Altman et al. (25). The mono- to the following formula: [1 Ϫ (ratio of unprimed cells/ratio of primed meric complexes were tetramerized by the addition of PE-labeled strepta- cells) ϫ 100], in which the ratio of unprimed cells is the percentage of vidin (BD Pharmingen) at a molar ratio of 4:1. CFSElow per CFSEhigh cells remaining in noninfected recipients, and the high Flow cytometry analysis ratio of primed cells is the percentage of CFSE cells remaining in in-

fected recipients. http://www.jimmunol.org/ The cells were incubated with saturating amounts of FITC-, PE-, Cy- Chrome-, and biotin-conjugated mAbs for 30 min at 4°C. To detect biotin- conjugated mAbs, cells were stained with CyChrome- or allophycocyanin- Statistical analysis ϩ conjugated streptavidin. For staining of epitope-specific CD8 T cells Data were analyzed by Student’s t test, and Bonferroni correction was d using the tetrameric H2-K -peptide complex, cells were incubated at 4°C applied for multiple comparison. A value of p Ͻ 0.05 was considered for 20 min in unconjugated-streptavidin (0.5 mg/ml; Sigma-Aldrich) and statistically significant. Fc block (2.4G2), followed by triple staining with FITC-CD44, Cy- Chrome-CD8␣, and PE-conjugated tetrameric H2-Kd-peptide complex (0.2–0.3 mg/ml) for 30 min at 4°C. The cells were analyzed using an Results FACSCalibur flow cytometer (BD Biosciences). ϩ CD153 is required for generation of long-lived memory CD8 T by guest on September 24, 2021 Analysis of intracellular cytokine synthesis cells Spleen cells were harvested from infected mice, washed, and suspended at We studied the kinetics of bacterial clearance and the expansion, 106 cells/ml in complete culture medium and then incubated for4hat37°C contraction, and stable memory of Ag-specific CD8ϩ T cells in in the presence of 10 mg/ml brefeldin A (Sigma-Aldrich) and 5 ␮g/ml CD153-deficient mice with a BALB/c background after i.p. inoc- LLO91–99 peptide. These cells were harvested, washed, and incubated for ϫ 5 30 min at 4°C with PE-conjugated anti-CD44 mAb and CyChrome-con- ulation with 1 10 CFU of L. monocytogenes strain EGD. We jugated CD8 mAb. After surface staining, cells were subjected to intracel- found that the numbers of bacteria increased to maximal levels on lular cytokine staining using a Fast Immune Cytokine System according to day 3 in the spleen and liver and thereafter cleared completely by the instructions of the manufacturer (BD Biosciences). The cells were day 10 after inoculation in both wild-type mice and CD153Ϫ/Ϫ ␮ washed and fixed in 100 l of FACS lysing solution (BD Biosciences) for mice. We also found that the bacteria were more rapidly eliminated 10 min at room temperature and were then washed again, resuspended in Ϫ/Ϫ 500 ␮l of FACS permeabilizing solution (BD Biosciences), and incubated in wild-type mice than in CD153 mice (data not shown). To for 10 min at room temperature. After washing, the cells were stained with directly follow the fate of the L. monocytogenes epitope-specific FITC-conjugated IFN-␥ mAb or FITC-conjugated isotype control rat IgG CD8ϩ T cells in CD153Ϫ/Ϫ mice after i.p. inoculation with L. (BD Pharmingen) for 30 min at room temperature, and the fluorescence of monocytogenes, we used intracellular IFN-␥ staining in response the cells was analyzed using a flow cytometer. to LLO91–99 peptide, the immunodominant epitope recognized by ϩ Cell culture H-2Kd-restricted CD8 T cells (Fig. 1A), and tetrameric MHC

ϩ molecule folding with the LLO91–99 peptide (Fig. 1B) for staining Magnetic bead-separated CD8 T cells (95% pure) from LN were cultured ϩ ␮ epitope-specific CD8 T cells. As shown in Fig. 1A, a significant in 200 l of complete culture medium in a 96-well flat-bottom plate (BD ϩ Biosciences) at density of 5 ϫ 105 cells/well with indicated concentrations number of CD8 T cells expressing high levels of CD44 in the ␥ of LLO91–99 peptide. To estimate IFN- production, the supernatant was spleen and peritoneal cavity were stained with intracellular IFN-␥ collected at 48 h. The IFN-␥ production in the supernatants was assayed in CD153Ϫ/Ϫ mice on day 7 after infection with L. monocytogenes using DuoSet ELISA development system (Genzyme). Proliferative activity of CD8ϩ T cells was assessed by incorporation of [3H]thymidine. The at the induction stage of effector T cells, albeit at a lesser level of cells were pulsed with [3H]thymidine 6 h before harvesting. [3H]Thymi- CD44 than that found in wild-type mice. However, the absolute ϩ dine incorporation was then determined by liquid scintillation counting. number of LLO91–99-specific CD8 T cells in the peritoneal cavity of CD153Ϫ/Ϫ mice was 3.1 Ϯ 0.3 ϫ 105 cells in CD153Ϫ/Ϫ mice, RT-PCR comparable to that found in wild-type mice of 3.4 Ϯ 0.4 ϫ 105 Nylon wool-enriched spleen T cells were incubated with appropriate dilu- cells (Fig. 2). These results suggest that the generation of effector d tions of FITC-conjugated anti-I-A , IgM, and biotinylated anti-DX-5, anti- CD8ϩ T cells normally occurs in CD153Ϫ/Ϫ mice following Lis- CD11c, and anti-␥␦ TCR mAbs, and washed twice in HBSS. The cells teria infection. were then incubated with anti-FITC microbeads, streptavidin microbeads, ϩ ϩ and anti-CD4 mAb microbeads for 15 min at 4°C. CD8 T cells were In contrast, the numbers of LLO91–99-specific CD8 T cells enriched to Ͼ90% by negative selection using LD-positive depletion col- were significantly higher in the peritoneal cavity on days 28 and 42 The Journal of Immunology 4629

FIGURE 1. CD153 is required for generation of long-lived memory CD8ϩ T cells. A, Kinetics of ϩ LLO91–99-specifc CD8 T cells in the peritoneal cavities and spleen of CD153Ϫ/Ϫ mice and wild-type mice after infection with L. monocytogenes. Cells were harvested from CD153Ϫ/Ϫ mice and wild-type mice infected with 1 ϫ 105 CFU L. monocytogenes, washed, and suspended at 106 cells/ml in a complete culture medium and were then incubated for5hat 37°C in the presence of 10 ␮g/ml brefeldin A and 5 ␮ g/ml LLO91–99 peptide. These cells were harvested, washed, and incubated for 30 min at 4°C with PE-conjugated anti-CD44 mAb, biotin-conjugated CD8 mAb, and then CyChrome-conjugated streptavidin. The cells were stained with FITC-conjugated IFN-␥ mAb for 30 min at room temperature, and ﬂuorescence of the cells was analyzed using a ﬂow cytometer. The analysis gate was set on CD8ϩ Downloaded from T cells. Each number in a quadrant indicates the percentage of each quadrant. Representative results from three separate experiments are shown. B, H2- d ϩ K /LLO91–99 tetramer-positive CD8 T cells in the spleens of CD153Ϫ/Ϫ mice and wild-type mice at 84 days after L. monocytogenes infection. For staining d

of tetrameric H2-K -peptide complexes, spleen cells http://www.jimmunol.org/ from CD153Ϫ/Ϫ mice and wild-type mice infected with 1 ϫ 105 L. monocytogenes 42 and 84 days previously were incubated at 4°C for 20 min in unconjugated streptavidin (0.5 mg/ml) and Fc block (2.4G2), followed by triple staining with FITC- CD44, CyChrome-CD8␣, and PE-conjugated tet- d rameric H2-K /LLO91–99 for 30 min at 4°C. The cells were analyzed using a FACSCalibur ﬂow cytometer, and then the analysis gate was set on CD8ϩ T cells. Each number indicates the percentage of H2- by guest on September 24, 2021 d ϩ K /LLO91–99-positive cells in CD8 T cells. Repre- sentative results from three separate experiments are shown.

after infection at a relatively early stage of memory T cell gener- day 42 after L. monocytogenes infection, at which time the number ation as compared with cells found in wild-type mice, whereas the of Ag-specific CD8ϩ T cells in CD153Ϫ/Ϫ mice was much more ϩ Ϫ Ϫ number of Ag-specific CD8 T cells in the spleen of CD153 / equal to that in wild-type mice. The cells were analyzed by four- mice did not differ from that in wild-type mice on day 28 and had color flow cytometory for the simultaneous expression of CD8, Ϫ/Ϫ decreased in CD153 mice on day 42 after infection (Fig. 1A). CD44, and CD62L on LLO -specific CD8ϩ T cells. The ma- ϩ 91–99 Notably, the numbers of LLO -specific CD8 T cells were ϩ Ϫ/Ϫ 91–99 jority (87%) of LLO91–99-specific CD8 T cells in CD153 significantly decreased in the spleen, LN, and peritoneal cavity of mice shown gated in Fig. 3A were of the CD62Llow phenotype, Ϫ/Ϫ CD153 mice on day 84 at a relatively late stage of memory T whereas only 13% were CD62Lhigh. In contrast, the memory cell generation compared with those in wild-type mice (Figs. 1 and CD8ϩ T cells from wild-type mice shown gated in Fig. 4A were 2; p Ͻ 0.01). These results suggest that absence of CD153 in vivo low ϩ referred as to CD62L population (32.2%) and majority does not affect generation of Ag-specific effector CD8 T cells but high high ϩ CD62L (67.8%) population. The proportions of CD62L hampers the generation of long-lived memory CD8 T cells after ϩ cells in the LLO -specific CD8 T cells in the spleen, LNs, L. monocytogenes infection. 91–99 and peritoneal cavity of CD153Ϫ/Ϫ mice were significantly lower ϩ Ϫ/Ϫ Impaired generation of memory CD8 TCM cells in CD153 than those in wild-type mice on day 42 after infection. mice after Listeria infection TCM cells exhibit higher proliferative ability than do TEM cells (4). To further confirm impairment of generation of T cells in The memory T cell compartment can be divided into T and T CM CM EM CD153Ϫ/Ϫ mice following Listeria infection, we next analyzed the cell subsets based on the expression of several cell surface mole- ϩ cules such as LN-homing receptors (3, 4). CD62L expression is functional properties of LLO91–99-specific CD8 T cells from the Ϫ/Ϫ useful in distinguishing between these two subsets because T spleens of wild-type and CD153 mice on day 42 after infection CM ϩ high low in in vitro response to LLO peptide. The ability of CD8 T cells are mostly CD62L , whereas TEM cells are CD62L (3, 91–99 Ϫ/Ϫ 4). We first examined the expression of CD62L on LLO -spe- cells from CD153 mice to produce the IFN-␥ was comparable 91–99 ϩ cific CD8ϩ T cells in spleens of wild-type and CD153Ϫ/Ϫ mice on to that of CD8 T cells from wild-type mice (Fig. 3B). However, 4630 ROLE OF CD30/CD30L IN MEMORY CD8ϩ T CELLS Downloaded from

ϩ http://www.jimmunol.org/ FIGURE 3. Impaired generation of memory CD8 TEM cells in CD153Ϫ/Ϫ mice after Listeria infection. A, CD62L expression on ϩ Ϫ/Ϫ LLO91–99-specific CD8 T cells in CD153 mice and wild-type mice infected with L. monocytogenes 42 days previously. LLO91–99-specific CD8ϩ T cells from CD153Ϫ/Ϫ mice and wild-type mice at 42 days after infection were costained for CD62L expression. Plots are gated on ϩ ϩ ϩ ␥ FIGURE 2. Kinetics of absolute numbers of LLO91–99-specific CD8 T CD8 LLO91–99 tetramer-positive (tet ) cells. B, IFN- production and ϩ Ϫ Ϫ cells in CD153Ϫ/Ϫ mice and wild-type mice following L. monocytogenes proliferative activity of CD8 T cells from CD153 / mice or wild- ϩ ␥ infection. The absolute numbers of LLO91–99-specific CD8 T cells were type mice infected with L. monocytogenes 42 days previously. IFN- ϩ Ϫ/Ϫ calculated by multiplying total cells by percentage of LLO91–99-specific production by CD8 T cells from CD153 mice and wild-type mice by guest on September 24, 2021 CD8ϩ T cells producing IFN-␥ in each organ. Representative results from at 42 days after infection separated using magnetic beads (95% pure) three separate experiments are shown, and data are expressed as mean Ϯ was assessed by ELISA following LLO91–99 peptide stimulation. Pro- ϩ Ϫ Ϫ p Ͻ liferative activity of CD8 T cells from CD153 / mice and wild-type ,ء .SD of five mice of each group from the representative experiment 0.05, significantly different from the value for wild-type mice. mice at 42 days after infection was assessed by incorporation of [3H]thymidine. The data are representative of three independent experiments using pooled cells from three CD153Ϫ/Ϫ or wild-type mice. only CD8ϩ T cells from wild-type mice were capable of prolifer- ating in response to LLO91–99 peptide (Fig. 3B). From these re- ϩ d sults, it is concluded that CD8 TCM cells are poorly generated in of tetrameric H2-K /LLO91–99-positive cells was significantly de- Ϫ Ϫ CD153 / mice following L. monocytogenes infection, although creased in the CD153Ϫ/Ϫ mice on day 84 after infection compared

TEM cells accumulated in nonlymphoid tissues at a relatively early with the number of cells in wild-type mice (Fig. 2). stage of memory T cell generation. To evaluate protective immunity against L. monocytogenes in wild-type and CD153Ϫ/Ϫ mice inoculated with Listeria 84 days Lack of CD153 causes a progressive loss in protective immunity previously, we tested the ability to control a lethal challenge of L. against reinfection with Listeria ϫ 6 monocytogenes (1 10 CFU). As expected, LLO91–99-specific As described, the numbers of long-lasting Ag-specific memory CD8ϩ T cells poorly expanded in CD153Ϫ/Ϫ mice compared with CD8ϩ T cells in both nonlymphoid and lymphoid tissues were those in wild-type mice on day 5 after a secondary challenge (Fig. greatly decreased on day 84 after Listeria infection. We examined 4B). There was no difference in the number of bacteria in the the functional relevance of this decrease by analyzing Ag-specific spleen and liver of between unimmunized CD153Ϫ/Ϫ and wild- CTL activity in vivo in CD153Ϫ/Ϫ mice on day 84 after Listeria type mice (Fig. 4C). However, consistent with the results of levels infection. This analysis was done by monitoring the specific erad- of memory CD8 T cells, there was a marked deficiency in the Ϫ/Ϫ ication of an adoptive transferred target population of LLO91–99- protection of CD153 mice against lethal challenge with L. pulsed splenocytes that had been differentially labeled with CF- monocytogenes (Fig. 4C). Collectively, CD153Ϫ/Ϫ mice poorly SEhigh so as to be distinguishable from a cotransferred reference generated long-lived memory CD8ϩ T cells, which were not able population (CFSElow). Fig. 4A shows that strong and specific cy- to lyse peptide-coated target cells and were not able to confer totoxic effectors were detectable in the spleens of infected wild- protective immunity against reinfection. Although TEM cells had type mice. In contrast, the numbers of CTLs in CD153Ϫ/Ϫ mice accumulated in the CD153Ϫ/Ϫ mice on day 42 after infection, the were greatly decreased on day 84 after infection with L. monocy- mice poorly generated long-lived memory CD8ϩ T cells at a later togenes (Fig. 4A). The loss of functional CTL activity in stage of infection. This result suggests that TCM cells may be im- CD153Ϫ/Ϫ mice might be results of quantitative, but not qualita- portant for generation of long-lived memory CD8ϩ T cells fol- tive, decrease by the Ag-specific CD8ϩ T cells because the number lowing Ag exposure. The Journal of Immunology 4631

CD30L signaling induces CCR7 expression in TEM ϩ We found that long-lasting CD8 TCM cells are poorly generated in Ϫ/Ϫ ϩ CD153 mice, whereas CD8 TEM cells accumulate in nonlymphoid tissues at a relatively early stage of memory T cell generation after L. monocytogenes infection. Furthermore, we previously found by DNA chip analysis that CD30 signaling up-regulates CCR7 mRNA (23, 24). These ﬁndings give rise to the possibility that CD30

signaling makes TCM cells by inducing expression of LN-homing receptors, including CCR7. To address this issue, we first examined changes in expression of LN-homing receptors of purified Ϫ ϩ ϩ CD62L CD44 CD8 TEM after CD30 stimulation. The purified Ϫ ϩ ϩ CD62L CD44 CD8 TEM cells were stimulated with plate-bound CD30 mAb for 24 h. We first examined the expression of CD62L on

the surfaces of TEM cells after anti-CD30 mAb cross-linking, but TEM cells did not express CD62L molecules after stimulation. We next examined gene expression of chemokine receptors, including CCR5,

CCR6, and CCR7, in the TEM cells stimulated with anti-CD30 mAb. As shown in Fig. 5 after anti-CD30 mAb cross-linking, the gene expression of CCR7 was greatly up-regulated and the gene expression of Downloaded from

CCR5 was down-regulated in TEM cells. These results suggest that CD30/CD30L signaling may play an important role in the generation of long-lived memory CD8ϩ T cells after Ag exposure by triggering

the expression of CCR7 in TEM cells to make them migrate and dif-

ferentiate into TCM cells in nonlymphoid organs. These results suggest that CD30/CD30L signaling plays an im- http://www.jimmunol.org/

portant role in the differentiation of TCM cells through induction of CCR7 mRNA expression.

Discussion In the present study, we found that CD30/CD30L signaling is in- ϩ volved in differentiation of long-lived CD8 TCM cells following ϩ exposure to a microbe. Although CD8 TEM cells transiently ac- Ϫ/Ϫ

cumulated in the nonlymphoid tissues of CD153 mice after by guest on September 24, 2021 ϩ Listeria infection, long-lived CD8 TCM cells, which exhibit strong proliferative ability and play a role in protective immunity, were generated in only small numbers in these mice following Listeria infection. These results suggest that CD30/CD30L signaling may play an important role in the generation of long-lived ϩ CD8 TCM cells after exposure to a microbe.

FIGURE 4. Lack of CD153 causes a progressive loss in protective immunity against reinfection with Listeria. A, Cytotoxic responses of CD8ϩ T cells in CD153Ϫ/Ϫ mice and wild-type mice at 84 days after L. monocytogenes infection. In vivo killing of LLO91–99 peptide-pulsed target cells labeled with CFSEhigh in uninfected mice, wild-type mice and CD153Ϫ/Ϫ mice at 84 days after infection was determined by monitoring the specific eradication of an adoptive transferred target population of LLO91–99-pulsed splenocytes that had been differentially labeled with CFSEhigh so as to be distinguishable from a cotransferred reference population (CFSElow). The number shown represents the percentage of specific killing to that of control CFSElow-labeled cells Ϯ ϭ ϩ (mean SD; n 5). B, Expansion of LLO91–99-specific CD8 T cells in CD153Ϫ/Ϫ mice and wild-type mice on day 5 after secondary challenge. Tetrameric H2-Kd-peptide complexes or intracellular cytokine staining of FIGURE 5. CD30L signaling induces CCR7 expression in TEM cells. ϩ Ϫ/Ϫ LLO91–99-specific CD8 T cells in CD153 mice and wild-type mice were Up-regulation of CCR7 expression in T cells in response to CD30 cross- ϩ EM examined by flow cytometry and analyzed by gating on CD8 T cells. C, linking. Purified CD62LϪCD44ϩCD8ϩ T cells were stimulated with Ϫ Ϫ EM Protection against lethal challenges with L. monocytogenes in CD153 / mice plate-bound anti-CD30 mAb (clone CD30.1) for 24 h. Gene expressions of and wild-type mice infected with L. monocytogenes 84 days previously. the chemokine receptors CCR5, CCR6, and CCR7 in T cells stimulated Ϫ Ϫ EM CD153 / mice and wild-type mice that had each been infected with 1 ϫ 105 with anti-CD30 mAb were examined by quantitative RT-PCR. First-strand CFU L. monocytogenes 84 days previously were each challenged with a lethal cDNA was synthesized with random primers. cDNA encoding chemokine dose of L. monocytogenes (1 ϫ 106 CFU), the numbers of bacteria in spleens receptors were analyzed by real-time PCR using a TaqMan PCR kit and an and livers were counted 2 days later. Each column and vertical bar represent ABI PRISM 7000 sequence detector thermal cycler according to the rec- p Ͻ 0.05, significant difference ommended protocol of the manufacturer (Applied Biosystems). Data of ,ء .mean Ϯ SD of five mice in each group between the values for wild-type CD8 to BALB/c and the values for immune gene expression were normalized on the basis of rRNA mRNA levels. Ϫ Ϫ CD153 / CD8 T cells to BALB/c. Representative results from three separate experiments are shown. 4632 ROLE OF CD30/CD30L IN MEMORY CD8ϩ T CELLS

ϩ Upon encounter with a pathogenic microbe, Ag-speciﬁc T cells It is notable that CD8 TEM cells transiently accumulated in proliferate and differentiate into activated effector T cells. Most of nonlymphoid tissues in CD153Ϫ/Ϫ mice at a relatively early stage the activated T cells die by apoptosis (31), but the few that survive of the memory phase. L. monocytogenes was eliminated both in become memory cells and persist for a long period of time, some- CD153Ϫ/Ϫ mice and wild-type mice in Ͻ10 days after infection, Ϫ/Ϫ times throughout the life of an animal (32–34). Memory is depen- but preferential accumulation of TEM cells in CD153 mice was dent on the amount of surviving T cells after primary TCR-medi- still observed on day 42 after infection with L. monocytogenes.It ated activation and presumably on escape from activation-induced is possible that TEM cells can survive in nonlymphoid tissues for cell death or starvation cell death by apoptosis (35). It has been several weeks in the absence of a relevant Ag or CD30L. Accord- recently reported that naive CD8ϩ T cells receiving prolonged or ing to the linear differentiation theory, CD30/CD30L signaling is strong stimulation of T cell receptors can differentiate into effector essential for conversion of TEM cells into TCM cells at least partly cells and survive as memory T cells by enhancing IL-15/IL-7 re- via induction of CCR7 expression. Memory T cells in nonlym- sponsiveness (36). TNF/TNFR superfamily is known to play im- phoid tissues may express CCR7 by CD30L/CD30 signaling and portant roles in generation of effector T cells as accessory mole- then migrate to lymphoid tissues with abundant expression of li- cules during an immune response (6). CD153 has been detected on gands for CCR7 such as secondary lymphoid-tissue chemokine. activated mouse DCs (16). Therefore, the CD153-CD30 pair may Because of the lack of CCR7 expression by CD30/CD30L signal- Ϫ/Ϫ contribute to T cell priming at dendritic cell-T cell interaction like ing, TEM cells may remain in nonlymphoid tissues of CD153

4-1BB and OX40 (37–39). In addition, CD153 is expressed on mice, resulting in preferential accumulation of TEM cells in the activated T cells and can support T cell expansion during T-T cell nonlymphoid tissues. Alternatively, according to a different lin- Downloaded from interaction (37). There are several lines of evidence that CD4 help eage theory, TEM cells may be preferentially generated in lym- is important for generation of memory CD8 T cells following mi- phoid tissues in the absence of CD30L and migrate to nonlym- crobial infection (40). Therefore, it is possible that defective den- phoid tissues due to the lack of CCR7 expression, resulting in Ϫ/Ϫ dritic cell and T cell help may account for the altered CD8 T cell accumulation of TEM cells in nonlymphoid tissues of CD153 responses in CD153Ϫ/Ϫ mice. However, the results of the present mice after infection. study indicate that the generation of effector CD8ϩ T cells seems CD30 has been reported to be preferentially expressed by Th2 to occur normally in CD153Ϫ/Ϫ mice at induction phase of effector and T cytotoxic type-2 (Tc2) cells (5, 14, 42–44), and blocking of http://www.jimmunol.org/

T cells after Listeria infection. Furthermore, the number of TEM CD30L/CD30 signaling has been reported to suppress the devel- cells increased, albeit transiently, after Listeria infection in CD30/ opment of Th2 cells and to enhance the development of Th1 cells CD30LϪ/Ϫ mice. CD30/CD30L signaling might not be essential at in vitro (45). These results suggest that CD30L/CD30 signaling ϩ least for the generation of effector CD8 T cells and CD8 TEM plays a role in the proliferation, cytokine secretion, and survival of cells after exposure to a microbe. Th2 cells, although controversy results have been reported (46).

Because both TCM and TEM cells can be generated during the Tc1 and Tc2 cells are known to mutually regulate in their differ- same immune response to L. monocytogenes, a key question is entiation, and Th1 cells have been reported to be subjected to

how these subpopulations are related to each other. Recent studies apoptosis weaker than Tc2 cells (7, 31) Therefore, it is alterna- by guest on September 24, 2021 have provided new insights into the lineage relationship between tively possible that Tc2 cells producing IL-4 are selectively sup- Ϫ/Ϫ TCM and TEM cells (1–3). There is evidence suggesting that TEM pressed after infection in CD153 mice, resulting in preferential cells can convert into TCM cells under appropriate in vivo condi- accumulation of memory Tc1 cells in nonlymphoid tissues at a tions (4). The numbers of TEM and TCM cells changed substantially relatively early stage of the memory phase. However, our results of over time and did so in a reciprocal manner, with the number of intracellular IFN-␥ FACS staining and tetramer staining revealed ϩ Ϫ/Ϫ TCM cells increasing and the number of TEM cells decreasing. that the number of Ag-specific CD8 Tc1 cells in CD153 mice Wherry et al. (4) investigated this possibility directly by transfer- did not increase at the peak of a primary response in CD153Ϫ/Ϫ ring purified populations of TEM cells or TCM cells into secondary mice. Furthermore, memory Tc2 cells were never detected in wild- recipients and examining the fate of the transferred cells. These type mice following Listeria infection (data not shown). Deviation researchers found that TCM cells retained their phenotype for at from Tc2 cells to Tc1 cells in CD30L-deficient mice may not be ϩ least 30 days after transfer, whereas half of the TEM cells acquired due to an increase in the number of memory CD8 TEM cells. the phenotype of TCM cells during this period. They therefore pro- The principal attribution of memory T cells is their ability to posed a linear differentiation model of a memory CD8ϩ T cell undergo cell division called “homeostatic proliferation” to subset. In the present study, the generation of TCM cells was im- maintain their number (1, 47). Homeostatic proliferation is paired in the absence of CD30L/CD30 signaling following Listeria thought to be required for the long-term maintenance of Ag- ϩ infection in vivo, and an LN-homing receptor, CCR7, was strongly driven memory CD8 T cells in vivo. A TEM cell population Ϫ low up-regulated in TEM cells after anti-CD30 mAb cross-linking in that is CCR7 CD62L has little homeostatic proliferative po- vitro. On the basis of the linear differentiation theory, our results tential, and this subset therefore does not seem to be a perma- suggest that CD30/CD30L signaling plays an important role in the nent memory population (4). In contrast, a TCM cell subset that generation of long-lived memory CD8ϩ T cells after exposure to is CCR7ϩCD62Lhigh is capable of efficient homeostatic prolif- an Ag by triggering differentiation into TCM cells via induction of eration and may survive for a long time in lymphoid tissues (4). ϩ the expression of CCR7 on TEM cells in nonlymphoid organs. In In the present study, memory CD8 T cells in both lymphoid contrast, in the case of CD8ϩ T cells, there is some evidence that and nonlymphoid tissues of CD153Ϫ/Ϫ mice decreased dramat-

TCM and TEM cells might be generated differentially during an ically at the late stage of infection. Most of the memory TEM immune response depending on the conditions of activation (5, cells in nonlymphoid tissues of CD153Ϫ/Ϫ mice might die by ϩ 41). On the basis of the different lineage theory that CD8 TCM apoptosis due to the lack of their responsiveness to survival and TEM cells are largely independent subpopulations, the possi- signals or survival signals themselves. Recent studies have sug- ϩ bility that fewer CD8 TCM cells are generated in the absence of gested that cytokines such as IL-15/IL-7 are involved in the CD30L/CD30 signaling at the time of immune response after pri- proliferation and survival of Ag-driven memory CD8ϩ T cells mary infection with L. monocytogenes cannot be excluded. in the absence of Ag (48–53). The Journal of Immunology 4633

The TNFR-associated factor (TRAF)-linked TNFR family an Ag by triggering differentiation into TCM cells via induction of members CD40, OX40, 4-1BB, and CD27 by virtue of their anti- expression of CCR7 on TEM cells in nonlymphoid organs. apoptotic effects, would be prime candidates to shape T cell memory (6). These molecules might be responsible for maintenance of Acknowledgments

TCM cells in the lymphoid tissues. It has recently been reported We thank K. Kaneda, Y. Kobayashi, and S. Takashima for technical that CD4ϩCD3Ϫ accessory cells have low levels of CD80 or CD86 assistance. expression but express high levels of the two TNF ligands OX40 ligand and CD153 in the B cell area of lymphoid tissues (16). Disclosures OX40 has been found to up-regulate expression of the CXC che- The authors have no financial conflict of interest. mokine receptor (CXCR) 5 and allows T cell migration into B cell References areas of peripheral lymphoid organs (54). We found that CD30 1. Kaech, S. M., E. J. Wherry, and R. Ahmed. 2002. Effector and memory T-cell stimulation also induced CXCR5 expression on TEM cells (our differentiation: implications for vaccine development. Nat. Rev. Immunol. 2: unpublished observation). Therefore, it is possible that CD30 sig- 251–262. 2. Lanzavecchia, A., and F. Sallusto. 2000. Dynamics of T lymphocyte responses: naling up-regulates the expression of CCR7 and CXCR5 on mem- intermediates, effectors, and memory cells. Science 290: 92–97. ory T cells, enabling these T cells to migrate to B cells areas of 3. Sallusto, F., D. Lenig, R. Forster, M. Lipp, and A. Lanzavecchia. 1999. Two lymphoid tissues. Survival signaling from CD4ϩCD3Ϫ accessory subsets of memory T lymphocytes with distinct homing potentials and effector functions. Nature 401: 708–712. cells in lymphoid tissues may play an important role in the main- 4. Wherry, E. J., V. Teichgraber, T. C. Becker, D. Masoqust, S. M. Kaech, R. Antia, U. H. von Andrian, and R. Ahmed. 2003. Lineage relationship and protective tenance of TCM cells for a long period. It is also possible that CD30 signaling provides direct survival signal or the responsiveness to immunity of memory CD8 T cell subsets. Nat. Immunol. 4: 225–234. Downloaded from ϩ 5. Weninger, W., M. A. Crowley, N. Manjunath, and U. H. von Andrian. 2001. memory CD8 T cells in addition to up-regulation of the expres- Migratory properties of naive, effector, and memory CD8ϩ T cells. J. Exp. Med. sion of homing receptors. CD30 uses TRAF molecules, particu- 194: 953–966. 6. Croft, M. 2003. Co-stimulatory members of the TNFR family: keys to effective larly TRAF2 and TRAF5, to induce downstream signals (55–57). T-cell immunity? Nat. Rev. Immunol. 3: 609–620. TRAF2 is thought to be responsible for NF-␬B activation and for 7. Hildeman, D. A., Y. Zhu, T. C. Mitchell, J. Kappler, and P. Marrack. 2002. Molecular mechanisms of activated T cell death in vivo. Curr. Opin. Immunol. the antiapoptotic effect mediated by CD30 (55–57). Although lym- 14: 354–359. Ϫ/Ϫ http://www.jimmunol.org/ phocyte homeostasis does not seem to be affected in TRAF5 8. Bourgeois, C., B. Rocha, and C. Tanchot. 2002. A role for CD40 expression on ϩ ϩ mice, TRAF2Ϫ/Ϫ mice are severely lymphopenic, suggesting that CD8 T cells in the generation of CD8 T cell memory. Science 297: 2060–2063. this molecule plays a role in T and B cell survival (58, 59). Anal- 9. Janssen, E. M., E. E. Lemmes, T. Wolfe, U. Christen, M. G. von Herrath, and ϩ ysis of the virus-specific response of CD4 T cells isolated from S. P. Schoenberger. 2003. CD4ϩ T cells are required for secondary expansion and memory in CD8ϩ T lymphocytes. Nature 421: 852–856. the spleen of immunized mice confirmed that CD30 is essential for 10. Shedlock, D. J., and H. Shen. 2003. Requirement for CD4 T cell help in gener- adequate expansion of Ag-activated T cells. It is still unclear ating functional CD8 T cell memory. Science 300: 337–339. whether CD153 expression is required for maintenance or emer- 11. Sun, J. C., and M. J. Bevan. 2003. Defective CD8 T cell memory following acute high infection without CD4 T cell help. Science 300: 339–342. gence of CD62L cells at the late stage after Listeria infection. 12. Du¨rkop, H., U. Latza, M. Hummel, F. Eitelbach, B. Seed, and H. Stein. 1993.

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