Programmed contraction of CD8+ T cells after infection - Nature

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Programmed contraction of CD8+ T cells after infection Vladimir P. Badovinac1, Brandon B. Porter2 and John T. Harty1,2 Published online: 28 May 2002, doi:10.1038/ni804

The extent of infection and rate of pathogen clearance are thought to determine both the magnitude of antigen-specific CD8 + T cell expansion and the ensuing contraction to a stable number of memory cells. We show that CD8 + T cell expansion after Listeria monocytogenes infection was primarily dependent on the initial infection dose or amount of antigen displayed, and was also influenced by the rate of pathogen clearance. However, the onset and kinetics of CD8 + T cell contraction after L. monocytogenes and lymphocytic choriomeningitis virus infections were independent of the magnitude of expansion, dose and duration of infection or amount of antigen displayed. Thus, major features of antigen-specific CD8 + T cell homeostasis, including the contraction phase of an immune response, may be programmed early after infection.

Results

In response to infections with viruses and intracellular bacteria, antigenspecific CD8+ T cells undergo massive expansion, followed by contraction to a stable number of memory cells1. Expansion is required to produce sufficient effector CD8+ T cells, which use cell-mediated cytolysis and cytokine production to defend the host against rapidly multiplying microbes2. The ensuing contraction phase, in which ∼90% of the effector T cells are eliminated, is essential to preserve flexibility in response to the universe of pathogenic microbes, while maintaining heightened defense against previously encountered pathogens. In many experimental systems, the onset of CD8+ T cell contraction is temporally correlated with pathogen clearance3, suggesting that the duration of infection may influence both the expansion and contraction of antigen-specific CD8+ T cells. The magnitude of CD8+ T cell expansion after experimental infection often depends on the number of microbes in the initial exposure. After only a brief period of antigen exposure, CD8+ T cells can undergo substantial expansion and differentiation into functional memory cells4–6. These data suggest that early events after infection may dictate the magnitude of CD8+ T cell expansion. However, higher initial infection may also result in delayed clearance of the pathogen, and it is not understood how the rate of microbial clearance influences the magnitude of CD8+ T cell expansion and the subsequent contraction phase. In addition, although a causal relationship between pathogen clearance and CD8+ T cell contraction is appealing, this relationship has not been established experimentally. After infection with an attenuated strain of Listeria monocytogenes that is cleared from both interferon-γ (IFNγ)–deficient and wild-type (WT) mice, IFN-γ–deficient mice show delayed contraction of antigen-specific CD8+ T cells compared to WT animals7. Thus, clearance of infection does not always result in CD8+ T cell contraction, suggesting that these events may not be linked. We addressed here the impact of pathogen clearance on CD8+ T cell expansion and contraction in murine models of infection (L. monocytogenes and LCMV) that allowed us to manipulate both the initial amounts and duration of infection.

Influence of expansion magnitude on contraction We determined whether the magnitude of expansion influences the onset and kinetics of antigen-specific CD8+ T cell contraction. BALB/c mice mount dose-dependent CD8+ T cell responses to amino acids (aa) 91–99 of listeriolysin O, LLO(91–99), and aa 217–225 of p60, p60(217–225), at day 7 after infection with an attenuated strain of L. monocytogenes (which is actA-deficient, median lethal dose (LD50) >107)7,8. BALB/c mice were infected with ∼105 (low dose) or ∼107 (high dose) actA-deficient L. monocytogenes, and splenic CD8+ T cell responses to LLO(91–99) and p60(217–225) were determined on days 5, 7, 8 and 11 after infection by intracellular cytokine staining for IFN-γ (Fig. 1a,b). Both infection doses elicited CD8+ T cell expansion; however, as early as 5 days after infection, high dose–infected mice contained about fivefold more antigen-specific CD8+ T cells. This result may reflect differences in the number of antigen-specific precursor CD8+ T cells recruited into the response5 (V. P. B. and J.T.H., unpublished data). By day 7, the number of antigen-specific CD8+ T cells in high dose–infected mice was more than tenfold that in low dose–infected mice (Fig. 1b). This was primarily due to a higher rate of expansion in the high dose–infected mice between days 5 and 7 (Fig. 1c). Thus, both the number of precursor cells recruited and the rate of expansion of the recruited cells may contribute to the magnitude of CD8+ T cell expansion. The peak of the response for both high and low dose–infected mice appeared to be day 7; the number of antigen-specific CD8+ T cells decreased uniformly in the spleen at day 8 after infection (Fig. 1b). This result was seen in the CD8+ T cell response to either epitope, despite differences in the magnitude and rate of CD8+ T cell expansion after infection with different doses of L. monocytogenes. In addition, the early kinetics of contraction (days 7 to 11) were identical in mice infected with ∼107 or ∼105 L. monocytogenes (Fig. 1b,c). Thus, the onset and early kinetics of antigen-specific CD8+ T cell contraction were independent of the magnitude of the expansion phase after

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Department of Microbiology and 2Interdisciplinary Graduate Program in Immunology, University of Iowa, Iowa City, IA 52242, USA. Correspondence should be addressed to J.T. H. ([email protected]). http://immunol.nature.com

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Figure 1. Relationship between CD8+ T cell expansion and contraction. BALB/c mice were infected with 0.8 × 107 (High) and 0.8 × 105 (Low) actA-deficient L. monocytogenes, then antigen-specific CD8+ T cells in the spleens were identified by intracellular IFN-γ staining after incubation with or without LLO(91–99) and/or p60(217–225). (a) Numbers represent the percentage of LLO(91–99) + p60(217–225)–specific CD8+ T cells from representative mice on various days after infection. Background (no peptide) was 50% of splenic CD8+ T cells after LCMV infection16,22). The contraction phase, therefore, is regulated in a precise fashion that may be independent of differences in pathogen biology. However, it is not known whether this relationship is preserved when the magnitude of CD8+ T cell expansion is altered after primary infection with various doses of the same attenuated pathogen or during the secondary response. We addressed these questions by measuring primary, memory and secondary responses of L. monocytogenes–specific CD8+ T cells after infection with various doses of actAdeficient L. monocytogenes (primary challenge) followed by a single high dose of virulent L. monocytogenes (secondary challenge) (Fig. 7). •

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Figure 8. Contraction of primary and secondary CD8+ T cell responses in the same host. BALB/c Thy1.1+ mice that received splenocytes from Thy1.2+ LCMV memory mice were challenged with 104 (∼1 LD50) virulent L. monocytogenes (strain XFL303) that expressed NP(118–126). Additional mice that did not receive splenocytes were infected with ∼0.1 LD50 of XFL303. Data are mean ± s.d. from three mice per group per time point. (a) Antigen-specific CD8+ T cells from representative mice on day 7 after XFL303 infection, as detected by intracellular cytokine staining. Samples were gated on CD8+ T cells; numbers represent the frequencies of Thy1.1+- (host) or Thy1.2+ (donor)-expressing CD8+ T cells in the spleen that were LLO(91–99)- or NP(118–126)-specific. Mice, with or without transferred Thy1.2+ cells, were analyzed for the following: (b–c) bacterial numbers in the spleen; (d–e) total numbers of Thy1.2+ NP(118–126)-specific (secondary), Thy1.1+ LLO(91–99)-specific (primary) or NP(118–126)-specific (primary) CD8+ T cells per spleen; and (f–g) normalized kinetics of antigen-specific CD8+ T cells that were undergoing primary and secondary responses. (h) Total number of Thy1.2+ NP(118–126)- and Thy1.1+ LLO(91–99)-specific CD8+ T cells per spleen in mice that received Thy1.2+ NP(118–126)-specific memory cells and were infected with 1 LD50 (with or without ampicillin treatment after 24 h) or 0.1 LD50 of L. monocytogenes strain XFL303. The 1 LD50 + ampicillin and 0.1 LD50 groups contained no detectable L. monocytogenes on day 5 after infection.


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peaks on day 5 after challenge21 (Fig. 7b). However, it appeared to contract more slowly, with ∼40% of antigen-specific cells remaining at day 11 after secondary challenge (6 days after the peak) (Fig. 7b,c), despite the substantial differences in starting number of memory cells, magnitude of expansion and control of infection (Fig. 7a). Therefore, the contraction of primary and secondary CD8+ T cell responses may occur with different kinetics, which is in general agreement with the secondary CD8+ T cell response after influenza infection of mice24.

Our data show a direct relationship between the magnitude of the primary CD8+ T cell response and the eventual number of memory cells (Fig. 7b). Mice that contained the fewest memory cells (low dose–immunized) mounted the most vigorous secondary response after challenge with virulent L. monocytogenes (Fig. 7c). This situation resulted in a secondary response that was very similar in total number of antigen-specific CD8+ T cells (less than twofold different in all groups), despite marked differences in the number of memory cells present at the time of challenge. These results were consistent with published studies of the CD8+ T cell response after oral L. monocytogenes infection23. In our experiment, ∼75% of antigen-specific CD8+ T cells were eliminated from the spleen between days 7 and 10 after primary infection, no matter how large the inoculum or magnitude of expansion (Fig. 7c). The secondary response to L. monocytogenes generally 624

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Expansion, contraction and memory Factors such as primed CD4+ T cells, inflammation and cytokine production, kinetics of pathogen clearance (Fig. 7a) or duration of antigen display may differentially contribute to the regulation of antigenspecific CD8+ T cell contraction in mice undergoing secondary versus •

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primary infection. To compare directly primary and secondary CD8+ T cell responses in the same host environment, we generated NP(118–126)-specific memory CD8+ T cells—which represented ∼15% of splenic CD8+ T cells (Web Fig. 2 online)—by LCMV Armstrong strain infection of BALB/c Thy1.2+ mice. Spleen cells from BALB/c mice were obtained 100 days after LCMV infection and transferred into congenic BALB/c Thy1.1+ mice, generating mice that contained ∼4×104 Thy1.2-derived NP(118–126)-specific memory CD8+ T cells per spleen (Web Fig. 2 online). These mice, and mice that did not receive memory T cells, were challenged with either 1.0 LD50 (∼104 CFU, with or without antibiotic treatment) or 0.1 LD50 of a virulent recombinant strain of L. monocytogenes (XFL303)25 that expresses both the LLO(91–99) epitope and the LCMV-derived NP(118–126) epitope. This approach allowed comparison of the expansion and contraction phases of the secondary (Thy 1.2+ NP(118–126)-specific memory cell) and primary (Thy1.1+ LLO(91–99) and NP(118–126)-specific cell) responses in the same host mice infected with the same pathogen (Web Fig. 2 online). The presence of NP(118–126)-specific memory cells decreased, but did not prevent a substantial primary response to both LLO(91–99) and NP(118–126) after infection with 1.0 LD50 of XFL303, compared to control mice infected with 0.1 LD50 (Fig. 8). This comparison was based on similar amounts of infection achieved in the different groups of mice (Fig. 8b,c). The onset and kinetics of contraction of the primary CD8+ T cell responses were essentially identical, whether or not the mice had NP(118–126)-specific memory cells (Fig. 8 d–g). In contrast, the secondary response of transferred NP(118–126) memory cells showed delayed contraction compared to the primary response to LLO(91–99) and NP(118–126) in the same host animals (Fig. 8 d–g), after infection with either 1.0 or 0.1 LD50 of XFL303 (Fig. 8h). In the latter experiment, antibiotic treatment 24 h after high-dose XFL303 infection did not alter the magnitude of expansion or onset of contraction in either the primary or secondary CD8+ T cell responses in the same host (Fig. 8h). Thus, the contraction of primary and secondary CD8+ T cell responses was differentially regulated; yet, in both cases, the onset of contraction appeared to be independent of infection dose or duration. The data are consistent with the suggestion that early events after infection determine the magnitude of both primary and secondary responses and instill a specific, yet different, program of contraction.

to antigen4–6. This response appears to be all-or-nothing, with all the activated CD8+ T cells progressing through at least eight divisions, the current limit of detection of the techniques used. One of these studies also suggested that the magnitude of CD8+ T cell expansion may be primarily dictated by the number of antigen-specific precursors recruited into the response, all of which undergo vigorous expansion5. Thus, it is possible to envision a system in which the onset of CD8+ T cell contraction is programmed to occur after a set number of divisions in each responding clone. This system would allow for coordinate contraction even when the magnitude of the response or clearance of infection differ, as the onset of contraction would be based on the number of divisions of each clone rather than require the population of responding cells to sense an environmental change such as pathogen clearance. Cell death or senescence is programmed by the number of divisions in many different eukaryotic cell types26. Resolving how this, or any other, control mechanism can precisely select one out of ten effector cells for survival into the memory pool remains an important goal in experimental immunology. What advantage would programmed contraction provide over a system in which CD8+ T cell contraction is triggered by clearance of infection? The most obvious situation in which this feature could be beneficial is in the case of persistent infection, as is seen in Pfp–/– mice after LCMV infection. In this scenario, the immune system mounts a significant CD8+ T cell response, whose magnitude is determined before any information as to the eventual outcome of infection has been received. If the massively expanded populations of antigen-specific effector CD8+ T cells do not resolve the infection, it is unlikely that continued expansion would be useful, and in fact may be detrimental to the host. Mortality during persistent LCMV infection of Pfp–/– mice that have the H-2b MHC has been observed by several groups and is dependent on the failure to exhaust antigen-specific CD8+ T cells20,27. Thus, programmed contraction and the eventual exhaustion of antigen-specific CD8+ T cells during persistent infection may reflect the need to minimize lethal immunopathology. Our data show that the contraction phases of primary and secondary CD8+ T cell responses are both independent of the rate of pathogen clearance. However, the onset and kinetics of contraction were clearly different in these populations, even in the same host animal, where variables such as CD4+ T cell help and inflammation were eliminated. The delayed contraction of secondary effector CD8+ T cells in WT mice is markedly similar to the protracted contraction phase of antigen-specific CD8+ T cells observed after primary L. monocytogenes infection of IFN-γ–deficient mice7. How might these two observations be related? IFN-γ signals through the constitutive IFN-γRα chain and an inducible IFN-γRβ chain28. Down-regulation of the β chain of IFN-γR has been observed in T helper 1 type CD4+ T cells29,30 and appears to play a role in the development of cytolytic activity in CD8+ T cells31. Impaired IFN-γ signaling—whether by receptor modulation or induction of inhibitory molecules in secondary, but not primary, CD8+ effector T cells—could account for the differential contraction of these populations. Efforts to determine the functional status of IFN-γR and signaling pathways in primary effector, memory and secondary effector CD8+ T cells are currently underway. Our data show that major features of CD8+ T cell homeostasis are minimally affected (magnitude of expansion) or unaffected (onset and kinetics of contraction) by alteration in the duration of infection or antigen display. Thus, early events after infection may instigate the majority of the antigen-specific CD8+ T cell program, a hypothesis with important implications for vaccine design, memory cell selection and the “window” of experimental focus32.

Discussion The current paradigm for CD8+ T cell responses is that both the expansion and contraction phases reflect the size of the inoculum, duration of infection and, by inference, antigen display. Although our results support a role for these factors in determining the magnitude of CD8+ T cell expansion, the data are not consistent with a model in which CD8+ T cells undergo contraction only when they “sense” the clearance of infection. Our latter conclusion is based on experiments demonstrating unaltered CD8+ T cell contraction under conditions in which the duration of infection and amount of antigen displayed were truncated from the onset of contraction (L. monocytogenes infection of WT mice) or extended past the onset of contraction (L. monocytogenes infection of WT mice and LCMV infection of Pfp–/– mice). Together, these data support a model in which the contraction of antigen-specific CD8+ T cells is hardwired to occur whether or not the host has successfully controlled the infection. How might CD8+ T cell contraction be programmed? TCR-transgenic CD8+ T cells undergo a program of substantial expansion and differentiation into memory cells after activation by a brief exposure http://immunol.nature.com

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Early programming of T cell populations responding to bacterial infection. J. Immunol. 165, 6833–6839 (2000). 14. Smith, G. A. et al. The two distinct phospholipases C of Listeria monocytogenes have overlapping roles in escape from a vacuole and cell-to-cell spread. Infect. Immun. 63, 4231–4237 (1995). 15. Kagi, D., Ledermann, B., Burki, K., Zinkernagel, R. M. & Hengartner, H. Molecular mechanisms of lymphocyte-mediated cytotoxicity and their role in imunological protection and pathogenesis. Annu. Rev. Immunol. 14, 207–232 (1996). 16. Murali, K. K. et al. Counting antigen-specific CD8 T cells: a reevaluation of bystander activation during viral infection. Immunity 8, 177–187 (1998). 17. Lau, L. L., Jamieson, B. D., Somasundaram,T. & Ahmed, R. Cytotoxic T-cell memory without antigen. Nature 369, 648–652 (1994). 18. Zajac, A. J. et al. Viral immune evasion due to persistence of activated T cells without effector function. J. Exp. Med. 188, 2205–2213 (1998). 19. 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Mice. BALB/c (which were Thy1.2+ and had the H-2d MHC) mice were from the National Cancer Institute (Frederick, MD). BALB/c Thy1.1+ mice, from R. Dutton (Trudeau Institute, Saranac Lake, NY) and Pfp–/– mice33 were maintained by brother-sister mating under specific pathogen–free conditions. Pathogen-infected mice were housed in the appropriate biosafety conditions. All mice were used at 8–16 weeks of age. Antibodies, peptides and tetramers. The following monoclonal antibodies were used. phycoerythrin (PE)-anti–IFN-γ (clone XMG 1.2, eBioscience, San Diego, CA), fluorescein isothiocyanate (FITC)–anti-CD8 or cychrome–anti-CD8 (clone 53-6.7, Pharmingen, San Jose, CA) and cychrome–anti-Thy1.2 (clone 53-2.1, Pharmingen). Synthetic peptides— which represented the defined L. monocytogenes LLO(91–99) and p60(217–225) H2Kd–restricted34,35 or LCMV NP(118–126) H-2Ld–restricted36 epitopes—were synthesized at the University of Iowa Protein Structure Facility. PE-conjugated Ld-NP(118–126) tetramers were from the NIH Tetramer Core Facility (Atlanta, GA). Bacteria and virus infection of mice. The virulent L. monocytogenes strain 10403s; XFL303 (which expresses the LCMV NP(118–126) epitope); and the attenuated L. monocytogenes strains DP-L1942 (which is actA-deficient), XFL303 (which is actA-deficient) and DP-L1936 (which is plcA and plcB–deficient) were resistant to streptomycin and were grown, injected and quantified as described8,10,25. In some experiments, ampicillin (2 mg/ml, Sigma, St. Louis, MO) was added to the drinking water 24 h after the initial L. monocytogenes challenge. CFU per spleen and gram of liver were determined on various days after infection as described10. The limit of detection (LOD) scores were as indicated. The Armstrong strain of LCMV (2 × 105 PFU/mouse intraperitoneally) was used as described7. Viral titers in homogenates of spleen, lung, liver and kidney were determined by plaque assay on VERO cells as described25. Adoptive-transfer experiments. BALB/c or Pfp–/– (Thy1.2+) mice were infected with LCMV Armstrong strain and at various days after infection, splenocytes were transferred into naïve BALB/c Thy1.1 mice. In some experiments, recipient mice were infected 1 or 2 days later with various numbers of XFL303 strain. At the indicated days after challenge or transfer, recipient mice were killed and spleens were taken for analysis. DEAD assay. The DEAD assay was done by mixing 5 × 104 CFSE-labeled antigen-specific CD8+ T cells10 with 1 × 106–5 × 106 splenocytes obtained at various times from L. monocytogenes–infected mice. Cells were incubated for 12 h in the presence of brefeldin A (Sigma) for the last 6 h of incubation. CFSE-labeled cells were analyzed by intracellular cytokine staining for IFN-γ production12. CD8+ T cells incubated with naïve splenocytes, in the presence or absence of synthetic peptides, served as positive and negative controls in the assay. Quantification of antigen-specific CD8+ T cells. The magnitude of the epitope-specific CD8+ T cell response was determined by intracellular cytokine staining for IFN-γ or MHC class I tetramer staining as described7. For intracellular cytokine staining, the percentage of IFN-γ+ CD8+ T cells in unstimulated samples from each mouse were subtracted from the peptide-stimulated value to determine the percentage of antigen-specific CD8+ T cells. The total number of epitope-specific CD8+ T cells per spleen was calculated from the percentage of IFN-γ+ CD8+ T cells, the percentage of CD8+ T cells in each sample and total number of cells per spleen. Note: Supplementary information is available on the Nature Immunology website. Acknowledgments We thank E. Gutierrez for technical assistance, the NIH Tetramer Core Facility for MHC class I tetramers and S. Perlman for critical review of the manuscript. Supported by the Leukemia and Lymphoma Society (V. P. B.) and NIH grants AI42767,AI46653,AI50073 (to J.T. H) and T32A07485 (to B. B. P.). Competing interests statement The authors declare that they have no competing financial interests. Received 13 March 2002; accepted 10 May 2002.

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