Functionally Heterogeneous CD8 T-Cell Memory Is Induced by Sendai Virus Infection of Mice

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1 JOURNAL OF VIROLOGY, Sept. 1999, p Vol. 73, No X/99/$ Copyright 1999, American Society for Microbiology. All Rights Reserved. Functionally Heterogeneous CD8 T-Cell Memory Is Induced by Sendai Virus Infection of Mice EDWARD J. USHERWOOD, 1 ROBERT J. HOGAN, 1 GRAHAM CROWTHER, 1 SHERRI L. SURMAN, 1 TWALA L. HOGG, 1 JOHN D. ALTMAN, 2 AND DAVID L. WOODLAND 1,3 * Department of Immunology, St. Jude Children s Research Hospital, Memphis, Tennessee ; Emory Vaccine Center, Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, Georgia ; and Department of Pathology, University of Tennessee Medical Center, Memphis, Tennessee Received 15 April 1999/Accepted 15 June 1999 It has recently been established that memory CD8 T cells induced by viral infection are maintained at unexpectedly high frequencies in the spleen. While it has been established that these memory cells are phenotypically heterogeneous, relatively little is known about the functional status of these cells. Here we investigated the proliferative potential of CD8 memory T cells induced by Sendai virus infection. High frequencies of CD8 T cells specific for both dominant and subdominant Sendai virus epitopes persisted for many weeks after primary infection, and these cells were heterogeneous with respect to CD62L expression (approximately 20% CD62L hi and 80% CD62L lo ). Reactivation of these cells with the antigenic peptide in vitro induced strong proliferation of antigen-specific CD8 T cells. However, approximately 20% of the cells failed to proliferate in vitro in response to a cognate peptide but nevertheless differentiated into effector cells and acquired full cytotoxic potential. These cells also expressed high levels of CD62L (in marked contrast to the CD62L lo status of the proliferating cells in the culture). Direct isolation of CD62L hi and CD62L lo CD8 T cells from memory mice confirmed the correlation of this marker with proliferative potential. Taken together, these data demonstrate that Sendai virus infection induces high frequencies of memory CD8 T cells that are highly heterogeneous in terms of both their phenotype and their proliferative potential. Virus-specific cytotoxic CD8 T cells (CTL) play a central role in the immune response to some virus infections by eliminating virus-infected cells (40). Control of the primary infection is followed by establishment of a memory population that is able to respond more rapidly and vigorously to a secondary infection with the same virus (12, 40). Recently, the development of major histocompatibility complex (MHC) class I tetramers has made it possible to directly identify antigenspecific T cells ex vivo (3). These studies have revealed that memory CD8 T cells are maintained at unexpectedly high frequencies in the spleen. For example, intraperitoneal infection of mice with lymphocytic choriomeningitis virus (LCMV) induces antigen-specific memory CD8 T cells at frequencies as high as 8% of splenic CD8 T cells (6, 29). This is much higher than the frequencies detected by classical limiting-dilution assays (LDA). Relatively high frequencies of CD8 memory T cells have also been detected following influenza virus infection (0.5% CD8 T cells) (16), although these frequencies are significantly lower than those induced by LCMV infection. Memory cells have classically been considered to have a resting phenotype; however, evidence is now emerging that some memory cells can respond rapidly to antigen and may maintain an effector function (4, 6, 19, 24, 29, 30, 32). Recent studies have revealed that there is substantial heterogeneity among populations of memory cells with respect to cell turnover (33) and phenotype as measured by activation markers such as CD62L and CD45RA/B/C (13, 33). In addition, there is evidence of functional heterogeneity in persistent LCMV * Corresponding author. Mailing address: Department of Immunology, St. Jude Children s Research Hospital, Memphis, TN Phone: (901) Fax: (901) Present address: Department of Biology and Biochemistry, The University of Bath, Bath BA2 7AY, United Kingdom. infection, where there is a population of antigen-specific CD8 T cells which express activation markers and proliferate in vivo but cannot exert an effector function (41). Other studies have suggested that a CD62L hi population of memory CD8 T cells which lose their immediate effector function can be induced in primary culture (30). However, these cells were generated following primary activation of T-cell receptor transgenic T cells in vitro and it is not clear how this relates to the situation with memory cells generated in response to a natural pathogen. Sendai virus is a type 1 parainfluenza which is a natural respiratory pathogen of mice. Intranasal infection of mice with Sendai virus elicits a potent CD8 T-cell response that is almost exclusively directed at a single K b -restricted nucleoprotein (NP) epitope defined by a nonapeptide, NP (9, 10, 20, 23). There is also a response to a subdominant epitope generated by the same NP peptide presented in the context of D b. Although CTL specific for the NP /D b epitope do not take part in the primary effector response, stable long-term memory for both NP /K b and NP / D b epitopes can be detected by LDA (8, 15). In the current study, we took advantage of MHC tetramers to further investigate the CD8 memory T cells elicited by Sendai virus infection of mice. The data show that Sendai virus induces a high frequency of memory CD8 T cells, which is much higher than that detected by LDA. Furthermore, there is significant heterogeneity among the CD8 memory T cells with respect to proliferative capacity, and this is associated with CD62L expression. MATERIALS AND METHODS Mice and virus. Female C57BL/6 mice were purchased from Jackson Laboratories (Bar Harbor, Maine) and housed under specific-pathogen-free conditions. Mice were infected at 6 to 12 weeks of age. The Enders strain of Sendai virus was grown, stored, and titrated as described previously (20). Mice were anesthetized by intraperitoneal injection with Avertin (2,2,2-tribromoethanol) and infected intranasally with % egg-infectious doses of Sendai virus. Influenza virus A/HK-x31 (H3N2) was grown, stored, and titrated as previously 7278

2 VOL. 73, 1999 CD8 T-CELL MEMORY FOR SENDAI VIRUS 7279 described (11). Mice were infected intranasally under anesthesia with 240 hemagglutination units of virus. Mice were considered to be memory mice when they had been infected with Sendai virus a minimum of 30 days previously. Peptides. Sendai virus NP and influenza virus NP (38) peptides were synthesized at the St. Jude Children s Research Hospital Center for Biotechnology by using a Perkin-Elmer Applied Biosystems (Berkeley, Calif.) 433A peptide synthesizer. Peptide purity was evaluated by reverse-phase high-pressure liquid chromatography analysis. Carboxyfluorescein (diacetate) succinimidyl ester (CFSE) labeling and culture conditions. Spleen cells were labeled with CFSE by incubation with 0.5 to 0.7 M CSFE diluted in Hanks balanced salt solution for 10 min in the dark. Cells were subsequently washed with Hanks balanced salt solution or complete tumor medium (22) before use. Unless otherwise stated, cultures were restimulated in vitro with NP peptide at a concentration of 0.5 g/ml and human recombinant interleukin-2 (IL-2) at 10 U/ml (R&D Systems, Minneapolis, Minn.) at a cell density of 10 6 /ml in 24-well plates. BrdU labeling. Spleen cells were restimulated in vitro with NP peptide as described above and cultured in the presence of 10 M BrdU and 1 M fluorodeoxyuridine FdUrd (14). After 48 h, cells were harvested and stained with NP /K b tetramer plus anti-cd8 and anti-brdu antibodies in accordance with established protocols (37). MHC tetrameric reagents and analysis. The construction of folded MHC class I-peptide complexes and their tetramerization have been described previously (29). Four tetramers were used. They were K b folded with Sendai virus NP (FAPGNYPAL), D b folded with the same peptide, and as controls, D b folded with influenza virus NP (ASNENMETM) and K b tetramers folded with the peptide TSINFVKI (p79) derived from murine gammaherpesvirus 68 (MHV-68) (35). Tetramers were stored as aliquots either at 70 C or at 4 C. The titer of the Sendai virus NP /D b tetramer was determined by using an NP /D b -specific CD8 T-cell line, and that of the Sendai virus NP /K b tetramer was determined by using bronchoalveolar lavage (BAL) fluid from Sendai virus-infected C57BL/6 mice. The influenza virus NP/D b peptide tetramer and murine MHV-68/K b acted as negative controls for the tetramers folded with Sendai virus epitopes; no cross-reactivity between the tetramers was detected. Staining with tetrameric reagent took place for 1 h at room temperature, followed by staining with anti-cd8 tricolor (Caltag, Burlingame, Calif.) and fluorescein isothiocyanate-conjugated anti-cd44 or anti-cd62l (Pharmingen, San Diego, Calif.) on ice for 20 min. For four-color staining of in vitro-restimulated cultures, CFSE-labeled cells were stained with allophycocyanin-conjugated anti- CD8 (Caltag), phycoerythrin-conjugated NP /K d tetramer, and biotinylated anti-cd44, anti-cd62l, anti-ly6c, anti-cd25, or anti-cd69 (Pharmingen), followed by phycoerythrin-cy7-conjugated streptavidin (Caltag). Three-colorstained samples were run on a Becton-Dickinson FACScan flow cytometer, and four-color-stained samples were run on a FACSCalibur. Data were analyzed by using CELLQuest software (Becton Dickinson Immunocytometry Systems, San Jose, Calif.). In some experiments, B cells were depleted before staining by panning on anti-immunoglobulin-coated flasks. Cell sorting. Cell preparations were stained with the NP /K b tetramer and/or antibodies as described above and then sorted into the appropriate cell populations by using either a FACSstar Plus or a MoFlo cell sorter (Becton Dickinson and Cytomation, respectively). Sorted cell populations were generally 90% pure. CTL assays. L-K b target cells have been described previously (7, 31). Cells were loaded with peptide and Na 51 CrO 4 as described previously (8). Briefly, monolayers of L-K b cells were incubated with 150 Ci of Na 51 CrO 4 with or without the NP peptide at approximately 300 g/ml in a minimal volume for 1 h. One milliliter of complete tumor medium was added, and the targets were incubated overnight. Cells were washed and counted, and standard protocols were used for 51 Cr release assays (2). The percentage of specific release was calculated by using the formula % specific release (experimental spontaneous)/(maximum spontaneous). Spontaneous release was typically 10% of maximum release. RESULTS High frequencies of memory CD8 T cells specific for Sendai virus epitopes are induced by primary Sendai virus infection. We have previously shown that the acute effector CTL response to Sendai virus infection in C57BL/6 mice is directed predominantly against a single NP-derived epitope, NP / K b (9). However, memory CTL precursors (CTLp) specific for both the dominant NP /K b epitope and a subdominant epitope involving the same peptide, NP /D b, are established following resolution of the primary infection (8). Interestingly, LDA indicates that CTLp specific for these two epitopes are induced at similar frequencies (approximately 0.05% CD8 T cells) despite their disparate contributions to the acute phase of the response. To further investigate the induction of CD8 T-cell memory for Sendai virus infection, we generated Sendai virus NP /K b and NP /D b tetramers to identify T cells with these antigen specificities directly. The specificities of these reagents were confirmed by using panels of T-cell hybridomas, and the tetramers were also shown to specifically stain T-cell lines or BAL fluid from acutely infected mice (16) (data not shown). Control tetramers folded with either influenza virus NP /D b or MHV-68 p79/k b did not stain T cells specific for these Sendai virus epitopes and have been described elsewhere (16, 34). Spleen cells were isolated from mice at various times after infection with Sendai virus and stained with Sendai virus-specific and control MHC tetramers plus CD8 and the activation markers CD44 and CD62L. As a control for these experiments, we also analyzed mice that had been infected with influenza virus. An unexpectedly high proportion (4 to 6%) of CD8 T cells from Sendai virus memory mice (but not control influenza virus memory mice) stained positive with the NP /K b tetramer, which detects T cells specific for the dominant epitope, at days 19 and 36 postinfection (Table 1). The memory NP / K b -specific T-cell frequency was reduced at 89 days postinfection but still relatively high at 2.1% of the total CD8 T cells. Despite the consistently high frequencies of memory CD8 T cells specific for the dominant epitope, detection of T cells specific for subdominant NP /D b was more variable and was, at most, one-half of that observed for NP /K b (2.2% versus 4% on day 36 postinfection; Table 1). This pattern was much more pronounced in the effector T-cell population isolated directly from the lungs of acutely infected mice, where 70.7% of the CD8 T cells stained with the NP /K b tetramer and 1.1% of the CD8 T cells stained with the NP /D b tetramer (Table 1) (9). Consistent with previous studies, a much smaller number of influenza virus NP /D b -specific CD8 T cells were detected in influenza virus-infected mice both in the acute infection (15.3% of CD8 T cells) and in memory (1.0% of total CD8 T cells and 2.3% of CD44 hi /CD8 T cells at 36 days postinfection) (16). Thus, the absolute frequencies of memory T cells induced by influenza virus and Sendai virus infections are substantially different despite the fact that LDA detects CTLp at similar frequencies from both types of infection. Phenotype of memory K b /NP specific CD8 T cells. We next investigated the expression of the activation-memory markers CD44 and CD62L on CD8 T cells specific for dominant NP /K b. All NP /K b -specific cells expressed high levels of CD44 (Fig. 1). Expression of CD62L was more heterogeneous, with the majority of NP /K b -specific cells being CD62L lo and approximately 20% being CD62L hi. Consistent with this finding, we noted a marked enrichment for NP /K b -specific T cells among the CD8 T cells with either a CD44 hi or a CD62L lo phenotype (Table 1). For example, at day 36 postinfection, 4% of CD8 T cells were NP /K b tetramer positive, compared with 28.7% of CD8 /CD62L lo cells. Interestingly, we did not see a similar enrichment of T cells specific for the subdominant NP /D b epitope among CD8 /CD44 hi or CD8 /CD62L lo T cells (Table 1). The reason for this difference is unclear but may be related to the subdominant status of this epitope. To determine whether the NP /K b -positive memory CTL were of uniform size, the forward scatter of the cells was examined. As shown in Fig. 2, we observed a discrete population of blasted NP /K b -specific cells among memory cells in the spleen. The proportion of NP /K b -specific cells with this blasted phenotype varied between 11 and 20%; however, this variation did not appear to be related to the length of time that the mice had been infected. CD62L and CD44 ex-

3 7280 USHERWOOD ET AL. J. VIROL. TABLE 1. Frequencies of CD8 cells specific for the dominant and subdominant Sendai virus epitopes as measured by MHC tetramer staining a Cell source (no. of days postinfection) and virus Total CD8 cells CD8 CD44 hi cells CD8 CD62L lo cells SV-NP/K b SV-NP/D b Flu-NP/D b SV-NP/K b SV-NP/D b Flu-NP/D b SV-NP/K b SV-NP/D b Flu-NP/D b BAL fluid (10) Sendai Influenza Spleen (19) Sendai Influenza Spleen (36) Sendai Influenza Spleen (89) Sendai a C57BL/6 mice were infected with either % egg-infectious doses of Sendai virus or 240 hemagglutination units of A/HK-x31. BAL fluid was analyzed 10 days postinfection, at the peak of the acute CD8 T-cell response. Spleen cells were analyzed at 19, 36, and 89 days postinfection to measure memory CTL frequencies. Staining was performed with MHC tetramers consisting of D b molecules folded with either the Sendai virus NP peptide (SV-NP/D b ) or the influenza virus NP peptide (Flu-NP/D b )ork b molecules folded with the Sendai virus NP peptide (SV-NP/K b ). Data are presented as percentages of tetramer-positive cells among total CD8, CD8 CD44 hi,orcd8 CD62L lo T cells. pression on these blasted cells was similar to that seen with the NP /K b -specific population as a whole (data not shown). Attempts to selectively sort these blasted memory cells for functional studies have thus far been unsuccessful. Taken together, these results suggested that the memory pool is a heterogeneous population consisting of cells in various stages of activation. Proliferative potential of memory NP /K b -specific CD8 T cells. Having established that the memory pool is heterogeneous with respect to activation-memory marker expression and cell size, we used an in vitro system to determine whether all of the cells in the memory pool had the same proliferative potential. In these and all subsequent experiments, mice were considered to be memory mice when they had recovered from an infection given at least 30 days previously. We took the approach of labeling cells with the fluorescent dye CFSE, which becomes progressively diluted out with each cell division (28). Spleen cells from Sendai virus-infected memory mice were removed, labeled with CFSE, and then restimulated in vitro with NP peptide and IL-2. The culture was then sampled each day and stained with an anti- CD8 antibody and the NP /K b tetramer, allowing us to monitor the division of NP /K b -specific cells in vitro. As shown in Fig. 3, the outgrowth of NP /K b -specific cells was easily detectable. In the first 2 days, there was little division of the tetramer-positive cells; however, these cells divided rapidly during the next 2 days and eventually expanded to account for approximately 66% of the CD8 T cells in the culture after 6 days. The NP /K b -specific cells divided in synchrony, and we did not observe discrete populations of cells which had undergone differing numbers of cell divisions, unlike those observed in primary T-cell responses in vitro (26, 30). Interestingly, we observed a population of NP /K b CD8 T cells that did not divide in vitro and remained CFSE hi. This could be seen most clearly at day 4 poststimulation (Fig. 3). At later time points, this population was less clear. This probably was due not to the disappearance of these cells but rather to dilution by the dividing cells in the culture. Both dividing and nondividing cells were alive and had high forward scatter, indicative of a blasted phenotype (data not shown). One possible explanation for the undivided population is that they were doublets consisting of a divided (CFSE lo ), tetramer-positive cell bound to a nondivided (CFSE hi ), nonspecific cell. To address this, we used the bandwidth feature of the FACScan to gate out all doublets. The nondivided, tetramer-positive cell population was not affected when doublets were excluded (data not shown), indicating that the data cannot be explained in this way. CFSE staining did not allow accurate quantification of the proportion of the original NP /K b -specific T cells that did not divide, since the cells had to pass through several rounds of division (and probably cell death) before there was a clear distinction between divided and nondivided cells. We therefore chose to use the incorporation of BrdU after short time peri- FIG. 1. Phenotype of NP /K b -specific CD8 cells during memory. Spleen cells from B6 mice at 36 days postinfection with Sendai virus were stained with anti-cd8, anti-cd44, and anti-cd62l antibodies plus tetrameric NP / K b. The upper graph shows CD8 and NP /K b staining with a lymphocyte gate, and the lower two graphs show activation marker expression gated only on NP /K b -specific CD8 cells. The data are representative of three experiments.

4 VOL. 73, 1999 CD8 T-CELL MEMORY FOR SENDAI VIRUS 7281 Downloaded from FIG. 2. Presence of blasted NP /K b -specific CD8 cells in the memory response. Spleen cells from C57BL/6 mice at various times (days [d]) after infection with Sendai virus were stained with anti-cd8 antibody plus tetrameric NP /K b. The graphs show forward scatter (FSC) and tetrameric NP /K b staining gated on all CD8 cells. Shown are the percentages of CD8 NP /K b cells in the quadrants. ods in culture to measure directly the number of cells starting to divide. We observed no significant BrdU incorporation after 24 h in culture (data not shown); however, after 48 h (when cell division is just beginning, as determined by CFSE staining [Fig. 3]) there was a clear population of cells that had incorporated BrdU (Fig. 4). The proportion of NP /K b -specific cells that remained BrdU negative was approximately 22% at this time point. Due to the rapid kinetics of cell division in vitro (Fig. 3), we could not exclude the possibility that a proportion of BrdU-positive cells had divided and thus skewed our calculations. We therefore consider the value of 22% to be a minimum estimate of the proportion of cells that did not divide. One possible explanation for this undivided population was that these cells had T-cell receptors with lower affinity for the NP /K b epitope. The low peptide concentration used in our experiments may not have been sufficient to transduce a signal through a low-affinity receptor. To exclude this possibility, we restimulated cells with progressively higher peptide doses and measured any change in the ratio of divided-toundivided cells after 4 days. The peptide concentration had little effect on the proportion of cells that failed to divide (data not shown). Even at peptide concentrations as high as 32 g/ ml, a sizeable fraction of NP specific cells remained CF- SE hi in culture. As this phenomenon was independent of the peptide concentration, we concluded that the nondividing cells were not merely those with low-affinity T-cell receptors. Function and phenotype of nondivided T-cell population. Previous studies have shown that restimulation of memory spleen cells from Sendai virus-infected mice with the NP peptide was a very effective method of generating effector CTL in vitro (8). We therefore wanted to test whether this effector CTL activity resided solely in the divided population or whether both the divided and nondivided populations gave rise to effector T cells. Thus, we stimulated CFSE-labeled memory cells with the NP peptide and separated the nondividing CD8 NP /K b CFSE hi population from the dividing CD8 NP /K b CFSE lo population 4 days later by fluorescence-activated cell sorting. Each population was tested for CTL activity on peptide-pulsed L-K b target cells. As shown in Fig. 5, both populations of cells were equally effective at lysing NP loaded L-K b target cells, indicating that the nondivided cells were fully functional effector CTL after in vitro restimulation. These data demonstrate that a significant fraction of antigen-specific memory CD8 T cells are able to differentiate into effector CTL in vitro without cell division. As mentioned above, there was heterogeneity in the expression of activation-memory markers in NP /K b -specific T cells in the spleens of memory mice. It was therefore important to test whether this heterogeneity extended to the phenotype of these cells after in vitro restimulation. We therefore used four-color flow cytometry to measure the expression of the T-cell activation markers CD25, CD44, CD62L, CD69, and on November 6, 2018 by guest

5 7282 USHERWOOD ET AL. J. VIROL. FIG. 3. Proliferation of NP /K b -specific CD8 T cells after in vitro restimulation with the NP peptide. Spleen cells from Sendai virus-infected memory mice were labeled with the fluorescent dye CFSE and then placed in culture with the NP peptide plus IL-2. Each day, a fraction of the culture was harvested and stained with anti-cd8 antibody plus either tetrameric NP /K b (left) or anti-t-cell receptor antibody (right). The graphs display data obtained by gating on all live, CD8 cells. The data are representative of three experiments. Ly6C on CFSE-labeled, NP /K b -specific, CD8 T cells in these cultures. As shown in Fig. 6, proliferating cells (CFSE lo ) were mostly CD44 hi CD62L lo CD69 lo and had a mixed phenotype with respect to CD25 and Ly6C. This was true irrespective of whether the cells were NP /K b specific (tetramer, right side) or nonspecific (tetramer, left side). The proliferation of nonspecific cells resulted from the presence of IL-2 (data not shown). In contrast, the nondividing, NP / K b -specific cells (CFSE hi tetramer ) were CD44 hi CD62L hi CD69 lo although they also had mixed CD25 and Ly6C phenotypes. As expected, the antigen-nonspecific population of nondividing cells was primarily CD44 lo and probably represents naive T cells. Thus, the key observation from this experiment is that the NP /K b -specific cells which had divided in vitro in response to peptide expressed a classical activated-memory phenotype (CD44 hi CD62L lo ) while the NP /K b -specific cells that did not divide were of the more unusual phenotype CD44 hi CD62 hi. Origin of nondividing cells. To establish which cells from the memory pool gave rise to the nondivided CD62L lo cells that we observed in vitro, we sorted different cell populations from the spleens of memory mice before CFSE labeling and in vitro restimulation with peptide. We sorted on the basis of CD44 and CD62L expression by using flow cytometry. Three cell populations were collected: CD44 hi CD62L hi, CD44 hi CD62L lo, and CD44 lo CD62L hi. There was no population corresponding to the other combination of markers (CD44 lo CD62L lo ). These cells were labeled with CFSE; mixed with unlabeled, uninfected splenocytes plus the NP peptide and IL-2; and then cultured for 4 days in vitro. Each culture was then stained with an anti-cd8 antibody and the NP /K b tetramer. As shown in Fig. 7, there was a marked difference in the ratio of divided-to-undivided cells in the various cell fractions. In the unsorted sample, the ratio of undivided-to-divided cells was 1:14.5 whereas the CD44 hi CD62L hi fraction gave a ratio of 1:2.5, demonstrating an enrichment for cells that did not divide in vitro. Conversely, the CD44 hi CD62L lo fraction gave an undivided-to-divided ratio of 1:18, indicating enrichment for cells that divide in vitro in response to peptide. The CD44 lo CD62L hi fraction did not contain a significant number of NP /K b -specific cells, as expected from previous results (Fig. 1). There was no exact correlation between CD62L expression and proliferative potential, since both divided and nondivided populations were present in cultures of CD62L hi and CD62L lo cells. Nevertheless, these data clearly show that the CD62L hi cells preferentially gave rise to cells with low proliferative potential whereas CD62L lo cells gave rise to cells with high proliferative potential. DISCUSSION The data presented here show that memory CD8 T cells specific for the dominant epitope are maintained at elevated levels for long periods after Sendai virus infection. This pop- FIG. 4. Enumeration of the nonproliferating fraction of NP /K b -specific CD8 cells. Spleen cells from Sendai virus memory mice were restimulated in vitro with the NP peptide plus IL-2 in the presence of 10 g of BrdU per ml plus 1 g of FdUrd per ml. A control culture received no BrdU-FdrUrd. Cultures were harvested after 48 h and stained with anti-cd8 and anti-brdu antibodies plus tetrameric NP /K b. Results show staining with anti-brdu antibody gated on all CD8 NP /K b lymphocytes. Similar data were obtained in two experiments.

6 VOL. 73, 1999 CD8 T-CELL MEMORY FOR SENDAI VIRUS 7283 FIG. 5. Both divided and nondivided NP /K b -specific CD8 cells possess an effector function. Spleen cells from Sendai virus memory mice were labeled with CFSE and then restimulated in vitro for 4 days with the NP peptide plus IL-2. Cells were then stained with anti-cd8 antibody plus tetrameric NP /K b prior to sorting by flow cytometry. The sample was gated to include only CD8 cells and then sorted into NP /K b CFSE lo (divided) and NP /K b CFSE hi (nondivided) cells. Sorted cells were then assayed for cytotoxicity in a 51 Cr release assay using target cells pulsed with NP (closed symbols) or no peptide (open symbols). E:T ratio, effector-to-target cell ratio. ulation was heterogeneous with respect to cell size and expression of the activation-memory marker CD62L. In addition, CD62L expression correlated with the ability of NP / K b -specific CD8 T cells to proliferate in vitro in response to antigen. This demonstrates that the memory CD8 T-cell pool consists of subpopulations with different functional capabilities that are associated with expression of CD62L. The high frequency of memory CD8 T cells induced by Sendai virus infection (2 to 6% of CD8 spleen cells) was surprising given that influenza virus, which induces a similar infection of the lung, only generates memory CD8 T cells on the order of 0.5 to 0.8% of CD8 spleen cells (16). In this regard, Sendai virus is more like LCMV, which induces antigen-specific memory cells up to 8% of the CD8 T cells in the spleen (6, 29). Importantly, these data demonstrate that not all respiratory infections generate low frequencies of memory CD8 T cells and suggest that LCMV and influenza virus may merely represent two ends of the spectrum. It is unclear what controls differences in magnitude between the memory CD8 T-cell pools induced by infections with different viruses. However, it has been suggested that the differences may be related to antigen load in lymphoid tissues, since LCMV directly infects the spleen whereas influenza virus only infects the lung. In support of this idea, Gallimore et al. have shown that the size of the immune response is proportional to the virus load in the LCMV system (17). However, the efficiency with which Sendai virus induces CD8 memory cells suggests that this is not the primary factor. Like influenza virus, Sendai virus replicates only in the lung and generates similar total viral loads. Live virus is not found in the spleen, and although antigenpresenting cells can migrate from the lung to the spleen, they are present at very low frequencies (39). It is possible that the amount of antigen carried to lymphoid tissue is larger in Sendai virus infection than in influenza virus infection; however, there is little information about the influenza virus system so a direct comparison is difficult. Interestingly, the acute effector responses to these two viruses are substantially different. Sendai virus infection induces a small inflammatory response in the lung, compared to the strong inflammatory response induced by influenza virus. However, the majority (70%) of CD8 T cells in the lungs of Sendai virus-infected mice are specific for the dominant epitope. This contrasts strongly with the relatively small fraction (15%) of dominant-epitope-specific CD8 T cells in influenza virus-infected mice. Even in a secondary immune response to influenza virus infection, only half of the CD8 T cells in the lungs are demonstrably specific for the dominant epitope (16). Thus, it is possible that the difference between the frequencies of antigen-specific memory cells established in these two systems simply reflects the CD8 T-cell composition of the prior acute response in the lung. We found previously that the frequencies of memory CD8 T cells specific for the dominant NP /K b and subdominant NP /D b epitopes were similar by using LDA (on the order of 1:2,000 CD8 T cells) (8). In contrast, more direct analysis of the dominance hierarchy among memory T cells using MHC tetramers reveals a pattern of immunodominance that mirrors that of the primary response. A lower frequency of subdominant-epitope-specific CD8 T cells has also been reported previously in listeria infection (5). This difference may relate to the ability of these cells to survive and proliferate in vitro, a necessary requirement for detection by LDA. We could clearly detect the proliferation of NP /D b -specific cells in vitro by using CFSE (data not shown); however, due to the small and variable population size it was not possible to make a meaningful comparison with the NP /K b response. The stimuli that perpetuate high frequencies of memory CD8 T cells are unknown. A role for persistent antigen cannot be ruled out (18), although several reports have argued against this idea (1, 21, 25). It has recently emerged that activated-memory cells (CD44 hi CD62L lo CD45RB hi ) turn over rapidly in vivo (36, 37), and this can be enhanced by the administration of type 1 interferon or IL-15 (36, 42). Sendai virus is a potent inducer of type 1 interferon; however, production presumably ceases in the lung after the infection is cleared. What triggers the synthesis of type 1 interferon or IL-15 during memory is unclear. We attempted to increase the pool of virus-specific memory CD8 T cells by administering IL-15 by the protocol used by Zhang et al. (42); however, we detected no change (data not shown). It is likely that this cytokine affects cell turnover and homeostasis (27) rather than the absolute size of the memory pool. We took advantage of the relatively high memory T-cell frequency to investigate functional differences within this population. Our experimental system is similar to that reported recently by Oehen and Brduscha-Riem (30), who studied the in vitro response of T-cell receptor transgenic T cells to an antigen from LCMV. These investigators showed that the memory CTL response consisted of two distinct populations: a CD62L lo population which could exert a rapid effector function and a CD62L hi population which required prolonged contact with target cells before developing an effector function. Our data

7 FIG. 6. Phenotype of divided and nondivided NP /K b -specific CD8 cells. Spleen cells from Sendai virus memory mice were labeled with CFSE and then restimulated in vitro for 4 days with the NP peptide plus IL-2. The culture was then stained with anti-cd8 antibody, tetrameric NP /K b, and antibodies recognizing lymphocyte activation markers. Gates were set to include live lymphocytes plus either CD8 NP /K b cells (left) or CD8 NP /K b cells (right). The data are representative of two experiments. 7284

8 VOL. 73, 1999 CD8 T-CELL MEMORY FOR SENDAI VIRUS 7285 FIG. 7. Proliferative potential segregates with the CD62L phenotype. Spleen cells from Sendai virus memory mice were stained with anti-cd44 and anti- CD62L antibodies and then sorted by flow cytometry into the three populations shown. Cells were labeled with CFSE and then added to an equal number of spleen cells from uninfected mice plus the NP peptide and IL-2 and cultured for 4 days. The cultures were then harvested and stained with anti-cd8 antibody and tetrameric NP /K b. The graphs show CFSE fluorescence and NP /K b staining gated on live CD8 lymphocytes. Similar data were obtained in two experiments. agree with this study and take it an important step further by extending the model to a natural pathogen infection of wildtype mice. We also show a functional segregation based on CD62L expression during memory. Our study demonstrates that the proliferative potential of antigen-specific CD62L lo cells is greater than that of CD62L hi cells. There is no absolute correlation between proliferative potential and CD62L expression, since we observed a population of cells derived from CD62L lo cells which failed to proliferate and a population of cells from CD62L hi cells which did proliferate. It will be interesting in future studies to elucidate whether these populations with altered proliferative potentials modulate CD62L expression accordingly. The relationship between the proliferative potential of memory CD8 T cells and their effector function is unclear. It is clear that Sendai virus-specific memory CD8 T cells are capable of rapid antigen responsiveness, as the frequency of cells which produce gamma interferon in response to 5 h of antigen stimulation is similar to that observed by using tetramer staining (39a). This is also true of other virus infections (16, 29). Other investigators have reported that the persistence of activated CD8 T-cell memory is necessary for protection from peripheral virus challenge (4, 24). In contrast, resting memory cells are sufficient to protect from systemic viral challenge but appear to lack the ability to recirculate into peripheral tissues. Further evidence for the persistence of effector cells is contained in the recent report of Selin and Welsh (32), who used sensitive target cells to show that cytolytically active CTL could be detected in the spleen for the lifetime of the mouse after LCMV challenge. These effector cells may be needed to eliminate virus rapidly from peripheral sites, whereas another population of memory cells takes longer to differentiate into effector cells and develops into a second wave of effector cells (4, 24). These latter cells may then be of importance in preventing systemic spread of the virus if the effector memory population fails to clear the virus. Our data showing that some memory T cells proliferate more than others lead to the speculation that these second-wave cells may be those that can proliferate and thus clonally expand to combat an infection. In contrast, the cells with low proliferative potential may comprise the first wave of effector cells which are able to attack infected cells more quickly. In conclusion, we used Sendai virus infection as a model system with which to address some of the issues concerning CD8 T-cell memory that had been demonstrated in other experimental systems. We showed that the memory CD8 T-cell population consists not of one homogeneous population but rather of subpopulations of cells with different phenotypes and capacities to proliferate in response to antigen. This has profound implications in terms of our understanding of immunological memory because it indicates that different memory CD8 T-cell populations perform different functions in vivo. 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