Rapid Development of Memory Phillip Wong, María Lara-Tejero, Alexander Ploss, Ingrid Leiner and Eric G. Pamer This information is current as J Immunol 2004; 172:7239-7245; ; of September 26, 2021. doi: 10.4049/jimmunol.172.12.7239 http://www.jimmunol.org/content/172/12/7239 Downloaded from References This article cites 30 articles, 12 of which you can access for free at: http://www.jimmunol.org/content/172/12/7239.full#ref-list-1

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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 © 2004 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Rapid Development of T Cell Memory1

Phillip Wong,* Marõ«a Lara-Tejero,* Alexander Ploss,*† Ingrid Leiner,* and Eric G. Pamer2*

Prime-boost is a promising strategy for inducing and amplifying pathogen- or tumor-specific memory CD8 T cell responses. Although expansion of CD8 T cell populations following the second Ag dose is integral to the prime-boost strategy, it remains unclear when, after priming, memory T cells become competent to proliferate. In this study, we show that Ag-specific CD8 T cells with the capacity to undergo extensive expansion are already present at the peak of the primary immune response in mice. These early memory T cells represent a small fraction of the primary immune response and, at early time points, their potential to proliferate is obscured by large effector T cell populations that rapidly clear Ag upon reimmunization. With sufficient Ag boosting, however, secondary expansion of these memory cells can be induced as early as 5Ð7 days following primary immuni- zation. Importantly, both early and delayed boosting result in similar levels of protective immunity to subsequent pathogen challenge. Early commitment and differentiation of memory T cells during primary immunization suggest that a short duration between priming and boosting is feasible, providing potential logistic advantages for large-scale prime-boost vaccination of human Downloaded from populations. The Journal of Immunology, 2004, 172: 7239Ð7245.

emory CD8 T cells are essential for immunity to many erate upon re-encountering Ag for 3 wk following priming (13). viral, bacterial, and protozoal pathogens (1). Upon pri- LCMV infection, however, induces extraordinarily large primary M mary activation by foreign Ag, CD8 T cells follow a immune responses (2, 3), and therefore may not reflect the type of program of proliferation and differentiation into CTL armed with T cell priming that occurs with vaccines. http://www.jimmunol.org/ effector functions that enable pathogen clearance or containment To explore the kinetics of memory development and the factors (2–8). After the expansion phase, the majority of Ag-specific CD8 that influence the magnitude of memory cell expansion in the set- T cells undergo programmed cell death, leaving a population of ting of a more typical CD8 T cell response, we examined mice memory CD8 T cells that swiftly proliferate upon secondary an- infected with the intracellular bacterial pathogen Listeria monocy- tigenic challenge. Substantial evidence suggests that CD8 T cells togenes. Peak CD8 T cell responses to L. monocytogenes infection transit through the effector phase before entering the memory pool occur 8 days following bacterial inoculation; subsequently, T cells (9, 10), but it remains unclear when, during the primary immune contract into stable memory populations within 14–21 days (14). response, effector T cells differentiate into long-lived memory In this study, we show that a subset of Ag-specific CD8 T cells can cells. A recent report showed that surface expression of the IL-7R undergo recall proliferative responses upon secondary encounter by guest on September 26, 2021 ␣-chain (IL-7R␣) distinguishes a small population of CD8 effector with Ag within 5–7 days of primary infection, even before the T cells at the peak of the primary response that gives rise to long- primary T cell response begins to subside. Interestingly, we find term memory cells (11); however, it remains unclear when, fol- that the rapid elimination of Ag by effector cells during an ongoing lowing priming, memory T cells acquire the ability to undergo immune response prevents the activation and proliferation of extensive proliferation in response to Ag. Although it was previ- memory cells, explaining the apparent lack of functional memory ously demonstrated that increased CD8 T cell proliferation can be CD8 T cells at early time points after primary immunization. The induced by additional Ag administration within 1 wk following ability of memory T cells to proliferate is revealed, however, when primary bacterial infection (12), a more recent report analyzing the the boosting dose of Ag exceeds the clearance capacity of effector CD8 T cell response to lymphocytic choriomeningitis virus T cells. Remarkably, boosting at early or late time points following (LCMV)3 infection concluded that memory T cells do not prolif- priming is similarly effective at enhancing both the size of sec- ondary memory T cell populations and protective immunity to sub- sequent pathogen challenge. These findings have significant im- *Infectious Diseases Service, Immunology Program, Department of Medicine, Sloan- plications for the design and delivery of prime-boost vaccines. Kettering Institute, Memorial Sloan-Kettering Cancer Center, New York, NY 10021; and †Immunology Program, Weill Graduate School of Medical Sciences of Cornell University, New York, NY 10021 Materials and Methods Mice, bacteria, and viruses Received for publication December 5, 2003. Accepted for publication March 25, 2004. BALB/cJ were obtained from The Jackson Laboratory (Bar Harbor, ME). The costs of publication of this article were defrayed in part by the payment of page BALB/c Thy-1.1-congenic mice were provided by C. Surh (Scripps Re- charges. This article must therefore be hereby marked advertisement in accordance search Foundation, La Jolla, CA). Wild-type (WT) L. monocytogenes strain with 18 U.S.C. Section 1734 solely to indicate this fact. 10403S, the ActAϪ/Ϫ strain of L. monocytogenes (15) (both provided by D. 1 This work was supported by National Institutes of Health Grants AI-39031 and Portnoy, University of California, Berkeley, CA), and the LLO92Ser mutant AI-42135, an Arthritis Foundation postdoctoral fellowship (to P.W.), and a Cancer strain of L. monocytogenes (mutation of the tyrosine in position 92 of Research Institute predoctoral fellowship (to A.P.). M.L.-T. is a Rosenwald Family/ listeriolysin to a serine (16)) were grown in brain-heart infusion (BHI) DeVaan-Irvington Institute postdoctoral fellow. broth (BD Biosciences, Sparks, MD). Vesicular stomatitis virus (VSV) 2 Address correspondence and reprint requests to Dr. Eric G. Pamer, Memorial Sloan- expressing the L. monocytogenes listeriolysin O (LLO) and p60 epitopes Kettering Cancer Center, 1275 York Avenue, New York, NY 10021. E-mail address: was generated by cloning a PCR-amplified fragment of the L. monocyto- [email protected] genes p60 protein encompassing the p60217–225 and p60449–457 epitopes 3 Abbreviations used in this paper: LCMV, lymphocytic choriomeningitis virus; into pVSV-XN2 (kindly provided by J. Rose, Yale University, New Haven, BHI, brain-heart infusion; LLO, listeriolysin O; VSV, vesicular stomatitis virus; WT, CT) using its unique XhoI and NheI sites. A 64-bp linker encoding wild type. LLO91–99 was then ligated in frame with the p60 fragment at the XhoI site

Copyright © 2004 by The American Association of Immunologists, Inc. 0022-1767/04/$02.00 7240 RAPID DEVELOPMENT OF T CELL MEMORY to generate a fusion LLO-p60 protein in pVSV-XN2. Recombinant VSV- immunization (13), an alternative explanation is that the secondary LLO-p60 viruses were then obtained, as previously described (17). Briefly, bacterial challenge is cleared with different kinetics at early and the LLO-p60-containing plasmid was transfected along with pBS-N, late time points following primary infection. Indeed, recent studies pBS-P, and pBS-L helper plasmids into baby hamster kidney cells infected with recombinant vaccinia virus expressing T7 polymerase (vTF7-3). Su- demonstrated that swift activation of Ag-specific T cells following pernatants were recovered 48 h later, spun down to remove debris, and primary infection rapidly eliminates APCs, thereby restricting ad- filtered through a 0.2-␮m filter to remove vTF7-3. The cleared supernatants ditional T cell priming (20, 21). To explore this possibility, we were used to infect baby hamster kidney monolayers. Supernatants were measured in vivo CTL activity using LLO -coated target cells recovered 48 h after the secondary infection, aliquoted, and stored at 91–99 Ϫ80°C. Viral titers were determined by plaque assay. at intervals after primary L. monocytogenes infection. Ag-specific Primary infections of mice were performed by injecting 2000 bacteria or cytotoxicity could be observed within 2 days of infection, consis- 5 ϫ 106 PFU of virus diluted in PBS into the tail vein. Viable bacterial tent with studies documenting the rapid development of CD8 T counts in infected mice were determined by homogenizing spleens in PBS cell effector function (18–20, 22) (Fig. 1b). In vivo cytolytic ac- containing 0.1% Triton X-100 and plating on BHI agar plates. tivity peaked 7 days after bacterial inoculation, corresponding with Peptides, Abs, tetramers, and flow cytometry the peak of CD8 T cell expansion, and subsequently waned to low, Peptides were synthesized by Research Genetics (Huntsville, AL). The but detectable levels 14–21 days after infection. To determine following Abs directed to mouse cell surface Ags were purchased from BD whether in vivo cytolytic activity correlated with Ag clearance, we PharMingen (San Diego, CA): anti-CD8a PerCP (53-6.7), anti-Thy-1.2 al- rechallenged mice with 106 bacteria 7, 14, or 21 days following lophycocyanin (53-2.1), and anti-CD62L allophycocyanin (MEL-14). Tet- primary infection and measured the number of viable L. monocy- d rameric H-2K /peptide complexes were generated, as described (12). Ap- togenes in spleens 24 h later. Mice rechallenged 2–3 wk after proximately 5 ϫ 105 to 1 ϫ 107 cells per sample were incubated on ice for ϳ Downloaded from 1 h with saturating concentrations of Abs and tetramers in FACS staining primary infection had 1000-fold more bacteria in spleens than buffer (PBS, 1% FCS, 0.05% sodium azide). Labeled cells were washed mice rechallenged 7 days following priming (Fig. 1c), supporting with FACS buffer, fixed in PBS containing 2% paraformaldehyde, and the notion that large numbers of activated effector T cells promote analyzed on a BD-LSR flow cytometer (BD Biosciences) using CellQuest rapid Ag clearance. or FlowJo (Tree Star, San Carlos, CA) software. CFSE labeling High dose Ag rechallenge within 1 wk of vaccination elicits secondary CD8 T cell expansion Splenocytes depleted of RBC were washed in PBS and resuspended at 5 ϫ http://www.jimmunol.org/ 107/ml in PBS containing 10 ␮M CFSE (Molecular Probes, Eugene, OR). To determine whether increasing the Ag dose at early time points After incubating at 37°C for 10 min in the dark, cells were immediately following primary immunization could overwhelm the clearance washed with cold RPMI 1640 medium (Life Technologies, Grand Island, capacity of the effector T cell response, and thereby uncover the NY) with 10% FCS before resuspending in PBS for injecting i.v. into mice. recall proliferative potential of Ag-specific T cells, we primed In vivo cytotoxicity assay mice with 2000 L. monocytogenes and rechallenged mice 7 or 90 4 5 6 Analysis of in vivo cytotoxicity was conducted similarly to the published days later with 2,000, 10 ,10,or10 bacteria. The expansion of protocol (18, 19). BALB/c splenocytes were divided into two populations LLO91–99-specific CD8 T cells was examined 5 days later. Al- and labeled with CFSE at a concentration of 3 ␮M (CFSEhigh)or0.3␮M though boosting with 2,000–10,000 bacteria 7 days after primary low high Ϫ6 (CFSE ). CFSE cells were pulsed with 10 M LLO91–99 peptide for by guest on September 26, 2021 low immunization did not induce significant expansion of Ag-specific 1hat37°C in the dark, while CFSE cells remained unpulsed. Subse- CD8 T cells, mice immunized 90 days previously demonstrated quently, CFSEhigh cells were washed and mixed with equal numbers of CFSElow cells before injecting 1.5 ϫ 107 total cells per mouse. Spleens significant secondary CD8 T cell expansion, even at the lowest from recipients were taken 15 h later for flow cytometric analysis to mea- rechallenge dose (Fig. 2a). Higher rechallenge doses (105–106 or- sure in vivo killing, as indicated by loss of the CFSEhigh Ag-pulsed pop- ganisms), however, resulted in significant (Ͼ8ϫ) secondary ex- ulation relative to the control CFSElow population. pansion of LLO91–99-specific CD8 T cells 7 days following prim- Adoptive transfers ing (Fig. 2a). Indeed, this booster effect was also seen in mice rechallenged with high doses of L. monocytogenes as early as 5–6 Splenocytes taken from naive or infected BALB/c mice were labeled with CFSE and injected i.v. into Thy-1.1 congenic BALB/c mice at 4–6 ϫ 107 days following primary infection (data not shown). These results cells/recipient. Ampicillin (Sigma-Aldrich, St. Louis, MO) was given to indicate that the capacity of primed, Ag-specific T cells to undergo donor mice at 2 mg/ml in their drinking water 5 days before sacrificing to memory-like expansion in response to a second encounter with Ag ensure that no viable bacteria were transferred along with prepared spleno- is present at early time points during the primary CD8 T cell cyte suspensions into naive recipients. Some recipients were infected 24 h later with 2000 WT L. monocytogenes, and expansion of donor cells, dis- response. tinguished by Thy-1.2 expression, was assessed by flow cytometry at var- To determine whether the efficiency of in vivo Ag clearance ious times after infection. inversely correlates with the extent of memory T cell expansion upon boosting, we rechallenged mice with 106 bacteria 7, 14, or 21 Results days following priming with L. monocytogenes. All mice fully Ag reintroduced during the primary response is rapidly cleared cleared bacteria 5 days following rechallenge when secondary To determine the kinetics of CD8 T cell memory generation during CD8 T cell expansion was measured, regardless of the time of bacterial infection, we primed naive mice with a sublethal dose of boosting. Although substantial LLO91–99-specific T cell expansion 2000 WT L. monocytogenes and then rechallenged mice 7 or 90 was seen in all groups, incrementally enhanced proliferation was days later with 104 bacteria. CD8 T cells specific for the immu- seen when rechallenge was delayed by 2 and 3 wk (Fig. 2b). Sim- d nodominant LLO91–99/H-2K epitope were quantified by MHC- ilar trends were noted for CD8 T cell responses to the Listeria d peptide tetramer staining 5 days after the second bacterial inocu- p60217–225 and p60449–457 H-2K epitopes (data not shown). Thus, lation. As expected, there was robust expansion of LLO91–99- as the efficiency of in vivo Ag clearance diminishes, the magnitude specific CD8 T cells after rechallenge of mice immunized 90 days of the memory T cell response to similar Ag doses increases. previously (Fig. 1a). In contrast, mice boosted 7 days after primary infection exhibited very little memory T cell expansion in com- Secondary effector responses inhibit memory CD8 T cell parison with nonboosted mice (Fig. 1a). expansion Although these results are consistent with the notion that mem- To determine whether rapid in vivo Ag clearance was also capable ory T cells require several weeks to develop following primary of inhibiting the expansion of long-term memory CD8 T cells, we The Journal of Immunology 7241

FIGURE 1. In vivo CTL activity after primary im- munization correlates with rapid clearance of reintro- duced Ag and diminished secondary CD8 T cell expan- sion upon early boosting. a, BALB/c mice immunized 7 or 90 days previously with 2000 WT L. monocytogenes were left unboosted or rechallenged i.v. with 104 bacte- ria. Spleens were harvested 5 days later, and expansion d of LLO91–99/H-2K -specific T cells was examined by flow cytometry. Density plots are gated on live CD8 T cells and are representative of two mice per group show- ing similar results. b, In vivo CD8 T cell cytolytic ac- tivity peaks 7 days after primary infection and gradually high wanes. Equal numbers of CFSE , LLO91–99 peptide- pulsed BALB/c splenocytes and CFSElow, nonpulsed BALB/c splenocytes were coinjected i.v. into BALB/c Downloaded from mice that were uninfected or infected previously with 2000 WT L. monocytogenes for the indicated number of days. In vivo cytolysis of donor cells was assessed 15 h later by flow cytometry. Histograms are gated on CFSEϩ splenocytes in recipient mice. Numbers at the top of each plot represent the percentage of CFSElow or CFSEhigh cells of total CFSEϩ donor cells recovered. http://www.jimmunol.org/ Results are representative of two mice per group yield- ing similar data. c, Ag reintroduced early after primary immunization is rapidly cleared. After primary immu- nization, mice were challenged 7, 14, or 21 days later with 106 WT L. monocytogenes, and the number of vi- able bacteria was determined 24 h later by plating spleens on BHI agar. Results represent the mean of three mice per group, and the SE is indicated. by guest on September 26, 2021

primed mice with WT L. monocytogenes and boosted them 3 mo target Ags expressed in one vector and boosting is conducted using 4 5 later with 0, 2000, 10 ,or10 L. monocytogenes LLO92Ser, a strain similar Ags expressed in a different vector, such as the consecutive that lacks the LLO91–99 epitope (16). Mice were then rechallenged use of DNA vaccines, followed by attenuated viral vectors ex- 5 days later with 105 WT L. monocytogenes to stimulate expansion pressing the same proteins (23–28). This approach generates po- of LLO91–99-specific memory CD8 T cells. We predicted that mice tent cellular immune responses compared with the use of a single boosted with L. monocytogenes LLO92Ser would generate second- vector and minimizes the effect of neutralizing Abs and T cell ary effectors specific for all bacterial epitopes except LLO91–99, responses induced during priming. Because residual effector re- and that this response would rapidly clear the subsequent rechal- sponses to primary immunization appeared to interfere with the lenge with WT L. monocytogenes, thereby preventing the activa- expansion of memory T cells upon rechallenge, we postulated that tion of LLO91–99-specific memory CD8 T cells. Consistent with early memory responses to LLO91–99 should be enhanced upon this prediction, immune mice that were not boosted with L. mono- rechallenge with an infectious agent that expresses this epitope, but cytogenes LLO92Ser exhibited massive secondary expansion of is otherwise antigenically distinct from L. monocytogenes.We LLO91–99-specific CD8 T cells after rechallenge with WT L. therefore generated a strain of vesicular stomatitis virus that monocytogenes (Fig. 3). In contrast, mice boosted with as little as expresses the L. monocytogenes LLO91–99 and subdominant

2000 L. monocytogenes LLO92Ser were unable to mount significant p60217–225 and p60449–457 epitopes (VSV-LLO-p60). After immu- secondary LLO91–99-specific CD8 T cell responses upon WT L. nizing mice with this virus, we rechallenged animals with attenu- monocytogenes rechallenge, supporting the notion that an ongoing ated ActA-deficient L. monocytogenes 7, 14, 21, or 28 days later

Listeria-specific effector response can also prevent the activation and measured expansion of LLO91–99-specific CD8 T cells. Using of long-lived Listeria-specific memory CD8 T cells. this heterologous prime-boost strategy, we detected nearly maxi- mal memory responses 7 days after priming (Fig. 4), demonstrat- Efficient induction of early memory CD8 T cell responses by ing not only that the capacity for memory expansion is present at heterologous prime-boost vaccination this early time point following CD8 T cell priming, but also that Recently described vaccination schemes use a heterologous prime- boosting with a heterologous vector can efficiently overcome boost strategy in which primary immunization is performed using effector responses that limit the induction of memory responses. 7242 RAPID DEVELOPMENT OF T CELL MEMORY

FIGURE 2. Secondary expansion of CD8 T cells can be elicited within 1 wk of primary immunization upon rechallenge with high doses of Ag. a, BALB/c mice immunized 7 or 90 days previously with 2000 WT L. monocytogenes were left unboosted or rechallenged i.v. with 2000, 104,105,or106 d bacteria. Spleens were harvested 5 days later, and expansion of LLO91–99/H-2K -specific T cells was examined by flow cytometry. Density plots are gated on live CD8 T cells and are representative of two mice per group. b, Magnitude of secondary CD8 T cell expansion incrementally increases with longer duration between initial priming and rechallenge. BALB/c mice infected 7, 14, or 21 days previously with 2000 WT L. monocytogenes were boosted with 6

Ⅺ f Downloaded from 10 WT L. monocytogenes. Total numbers of LLO91–99-specific T cells from nonboosted ( ) and boosted ( ) mice were determined 5 days later by flow cytometry. Results are representative of two mice per group.

Early and late memory CD8 T cells exhibit similar expansion Pretransfer analysis of CD8 T cells from day 8 infected donors low kinetics upon secondary encounter with Ag in adoptive transfer showed a substantial population of CD62L LLO91–99-specific recipients effectors, while only a small number of LLO -specific memory 91–99 http://www.jimmunol.org/ high Secondary CD8 T cell expansion following early Ag re-exposure CD8 T cells, mostly CD62L , were found in day 46 infected might reflect a large number of effector T cells undergoing a few donor mice (Fig. 5b). No detectable division of donor T cells was additional rounds of proliferation or, alternatively, a smaller subset observed 2 days after infection of recipient mice, but by days 4 and of the responding T cells undergoing extensive proliferation. To 6, similar frequencies of expanded LLO91–99-specific CD8 T cells differentiate between these alternatives, we adoptively transferred were detected among day 8 and 46 infected donor T cells (Fig. 5b). CFSE-labeled splenocytes from naive mice or mice infected 8 vs The mean CFSE fluorescence intensities among the expanded cells 22 days previously with L. monocytogenes into naive Thy-1.1 con- were similar, indicating that cells from both types of donors di- genic mice, and examined the expansion of transferred, Ag-spe- vided at similar rates. Plotting the absolute number of LLO91–99- cific T cells after infection of recipient mice. In the absence of specific donor CD8 T cells that expanded from day 8 and 46 in- by guest on September 26, 2021 infection, donor CD8 T cells from all groups of mice did not divide fected mice over time demonstrated nearly identical expansion (Fig. 5a). Five days after bacterial infection, there was no detect- kinetics (Fig. 5c). Thus, despite the initial disparity in the number able expansion of CD8 T cells from naive donors, but CD8 T cells of Ag-specific CD8 T cells at the time of transfer, secondary ex- from day 8 and 22 immune mice had extensively proliferated, as pansion of donor CD8 T cells from recently or remotely immu- reflected by loss of CFSE, giving rise to remarkably similar pop- nized mice was similar, indicating that the T cell populations that ulations of LLO -specific CD8 T cells. Expansion of LLO underwent proliferation in response to Ag re-exposure were sim- 91–99 91– ilar in size and responsiveness. This result is most readily ex- 99-specific T cells was Ag driven and not due to nonspecificin- flammation, because infection of recipients with L. monocytogenes plained by similarly small subsets of memory T cells in both LLO stimulated secondary expansion of CD8 T cells from groups undergoing extensive division. The similar extent of CFSE 92Ser dilution in responding T cell populations demonstrates that they immune donors without generating LLO91–99-specific CD8 T cells. To compare the kinetics of memory T cell expansion, we trans- have undergone cell division a similar number of times. On the ferred CFSE-labeled immune splenocytes from day 8 or 46 in- basis of CFSE intensity, we estimate that responding T cells in ϳ fected mice into naive mice and measured donor CD8 T cell ex- both experimental groups have undergone 7 divisions. The sim- pansion at 2, 4, and 6 days following recipient infection. ilar size of the responding populations suggests that the starting populations were also of similar size. Assuming that cell loss or migration of cells into or out of the spleen was negligible during division, we estimate that 200–500 cells from day 8 infected do- nors and 600–800 cells from day 46 infected donors gave rise to the expanded memory T cell populations.

Memory cells generated from early or delayed boosting exhibit similar contraction kinetics and are long-lived To assess whether early and delayed boosting give rise to similarly FIGURE 3. Reduced memory expansion in the presence of secondary stable secondary memory CD8 T cell populations, mice vaccinated effectors. Mice immunized 95 days previously with 2000 WT L. monocy- 4 5 with VSV-LLO-p60 either 7 or 28 days previously were chal- togenes were boosted with 0, 2000, 10 ,or10 LLO92Ser L. monocytogenes Ϫ/Ϫ and rechallenged 5 days later with 105 WT L. monocytogenes. Expansion lenged with ActA L. monocytogenes, and the LLO91–99-spe- d cific CD8 T cell population was tracked over time. Mice boosted of LLO91–99/H-2K -specific T cells was examined by flow cytometry an- other 5 days later. Density plots are gated on live CD8 T cells and are at day 7 or 28 postimmunization exhibited substantial memory representative of two mice per group. The first plot on the left shows responses 5 days later, with ϳ2-fold greater numbers of Ag-spe- splenic LLO-specific CD8 T cells in nonboosted immune mice. cific T cells in mice boosted at day 28 relative to day 7 boosted The Journal of Immunology 7243

FIGURE 4. Heterologous prime-boost vaccination demonstrates that memory CD8 T cells are generated rapidly. BALB/c mice infected 7, 14, 21, or 28 ϫ 6 6 days previously with 5 10 PFU VSV-LLO-p60 were challenged with 10 attenuated ActA-deficient L. monocytogenes. Expansion of LLO91–99-specific CD8 T cells was determined 5 days later by flow cytometry. a, Density plots from nonboosted (top panels) and boosted (bottom panels) mice are gated on live CD8 T cells and are representative of two animals per group. b, Graph shows total numbers of splenic LLO91–99-specific CD8 T cells in nonboosted (Ⅺ) and boosted (f) mice challenged at the indicated day after primary infection. Downloaded from animals (Fig. 5d). The expanded memory populations contracted within 5–7 days of primary infection, and can be stimulated to similarly to ϳ12–13% of the peak response after 1 mo, with Ag- undergo rapid and robust expansion upon early secondary encoun- specific CD8 T cells persisting at levels well above those of non- ter with Ag. This is observed in priming and boosting with the

boosted animals. Thus, early and delayed boosting both generate same pathogen and even more dramatically in heterologous prime http://www.jimmunol.org/ significant numbers of long-lived, Ag-specific memory CD8 T boosting using viral and bacterial vectors. Importantly, early mem- cells. ory expansion does not reflect the stimulation of large numbers of effector T cells to undergo a few additional rounds of division, but Early and delayed boosting generate similar protective immunity rather reflects the extensive proliferation of a small subset of T To determine whether early and delayed boosting induced similar cells embedded within the greater responding T cell population. levels of protective immunity, we immunized mice with 2000 WT Our results are consistent with recent data indicating that memory L. monocytogenes, boosted animals 1 or 4 wk later with VSV- cell precursors can be found amid the primary response (11). How- LLO-p60, and then challenged both groups of mice 1 mo later with ever, we show that these cells already have the capacity to undergo 5 ϫ 104,105,or5ϫ 105 WT L. monocytogenes. As controls, we secondary expansion upon early Ag re-encounter. Further evidence by guest on September 26, 2021 also challenged naive mice and mice that were immunized 1 mo for these cells being bona fide memory T cells is provided by our previously with 2000 WT L. monocytogenes, but not boosted with data showing that these expanded cells provide robust long-term VSV-LLO-p60. As expected, naive mice exhibited progressively protective immunity, because both early and delayed boosting elic- increasing numbers of bacteria in the spleen 24–72 h after infec- ited similar levels of protection that were significantly higher than tion, and those given the highest dose of L. monocytogenes died by that of nonboosted mice when mice were challenged with the same day 3 (data not shown). In contrast, nonboosted, immune mice pathogen 1 mo later. appeared healthy and had lower, albeit significant, numbers of bac- We propose that, soon after infection, a subset of activated Ag- teria 24 h after challenge with the three doses of L. monocytogenes, specific effector CD8 T cells is selected for the long-lived memory but these organisms were gradually cleared over the subsequent 2 pool and rapidly acquires the potential to undergo extensive ex- days (Fig. 6). Remarkably, mice immunized with L. monocyto- pansion upon secondary Ag encounter. Such diversification would genes and boosted 7 or 28 days later with VSV-LLO-p60 both be consistent with the recent finding that CD4 effector T cells showed markedly increased protective immunity relative to non- responding to influenza infection exhibit broad heterogeneity in boosted immune mice, as reflected by the sharply reduced numbers functional capacity (29). Our results contrast with the slower rate of bacteria that could be cultured from the spleen at all times fol- of memory T cell development recently reported using the LCMV lowing L. monocytogenes challenge. Although slightly enhanced system (13), perhaps due to the unusually large number of effector protective immunity at 24 h postchallenge was observed after de- CD8 T cells generated during primary LCMV infection (2, 3), layed vs early boosting with high doses of L. monocytogenes chal- which would rapidly eliminate any booster Ag introduced at early lenge, it was clear that both early and delayed boosting success- times after immunization and prevent the efficient stimulation of fully conferred several logs of protection early after challenge the small number of functional memory cells that may already be relative to unboosted animals and allowed mice to clear a subse- present. Furthermore, increasing the priming dose of LCMV can quent lethal L. monocytogenes challenge much more rapidly than lead to clonal CD8 T cell exhaustion (30), suggesting that the nonboosted mice. Thus, the timing of the boost did not alter its massive CTL response to this virus may not be representative of ability to enhance the memory responses of challenged mice, pro- the type of CD8 T cell response elicited by most currently inves- viding strong support for the functionality of memory T cells ex- tigated vaccines. panded at early times after primary immunization. Our finding that increased doses of Ag can induce secondary T cell expansion early after primary immunization suggests that in Discussion vivo Ag presentation may be extended by increased doses of Ag. Understanding when develops is impor- This is an interesting possibility because we and others (20, 21) tant for the rational design of vaccines. Our findings indicate that recently showed that in vivo functional Ag presentation to prime T long-lived, Ag-specific memory CD8 T cells are already formed cells actually occurs within only a brief period of ϳ2–3 days after 7244 RAPID DEVELOPMENT OF T CELL MEMORY Downloaded from http://www.jimmunol.org/

FIGURE 5. Early and delayed re-exposure to Ag induces similar secondary expansion of CD8 T cells and contraction into stable long-lived memory populations. a, CFSE-labeled BALB/c splenocytes from naive mice or mice infected 8 vs 22 days previously with 2000 WT L. monocytogenes (Lm) were ϩ/ϩ transferred into Thy-1.1 BALB/c congenic hosts. Recipients were left uninfected or infected 24 h later with 2000 WT Lm or LLO92Ser Lm. Plots show ϩ ϩ expansion of LLO91–99-specific donor cells, as assessed by flow cytometric analysis of CFSE dilution among gated CD8 Thy-1.2 T cells 5 days postinfection. Results represent two mice per group. b, CFSE-labeled BALB/c splenocytes from mice infected 8 or 46 days previously with 2000 WT Lm ϩ/ϩ were transferred into Thy-1.1 BALB/c congenic hosts, which were infected 24 h later with 2000 WT Lm. Pretransfer analysis of the frequency of by guest on September 26, 2021

LLO91–99-specific CD8 T cells among donor splenocytes is shown on the left panels. Right panels, Show expansion of LLO91–99-specific donor cells in recipients at 2, 4, and 6 days postinfection, as assessed by flow cytometric analysis of CFSE dilution in gated CD8ϩThy-1.2ϩ T cells. Frequency and mean fluorescence intensity (in parentheses) of expanded cells are indicated in plots. Results represent two mice per group. c, Graph depicts the total number of

LLO91–99-specific donor CD8 T cells from day 8 infected (squares) vs day 46 infected (diamonds) donors over time in recipients that were not infected (open symbols) or infected (filled symbols) with 2000 WT Lm. d, Mice infected 7 days (squares) vs 28 days (circles) previously with VSV-LLO-p60 were 6 Ϫ/Ϫ either left unboosted (open symbols) or challenged with 10 ActA Lm (filled symbols), and the total number of LLO91–99-specific CD8 T cells was assessed at 5 and 35 days after the time of boosting. infection, due to swiftly acquired CTL activity that eliminates pro- cause we and other groups have shown that T cell responses are fessional APC. Higher Ag doses might lengthen this period of Ag largely programmed within the first 24 h of Ag encounter, resulting presentation, but this may be unnecessary for T cell priming be- in multiple rounds of Ag-independent proliferation over several

FIGURE 6. Early and delayed boosting generate similar levels of protective immunity. Mice immunized with 2000 WT L. monocytogenes were boosted 7 or 28 days later with 5 ϫ 106 PFU VSV-LLO-p60. Mice were then challenged 30 days later with 5 ϫ 104,105,or5ϫ 105 WT L. monocytogenes, and the number of viable bacteria in mice was determined 24, 48, and 72 h later by plating spleens on BHI agar. As controls, naive mice and mice immunized with 2000 WT L. monocytogenes 30 days previously, but not boosted, were also inoculated with the same challenge doses of WT L. monocytogenes. Results represent the mean of three mice per group, and the SE is indicated. The experiment was performed twice and yielded similar results. The Journal of Immunology 7245 days (5–8). However, it is tempting to speculate that extended Ag 9. Opferman, J. T., B. T. Ober, and P. G. Ashton-Rickardt. 1999. 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