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The Journal of Immunology, 2004, 172: 7239-7245.
Copyright © 2004 by The American Association of Immunologists

Rapid Development of T Cell Memory1

Phillip Wong*, María Lara-Tejero*, Alexander Ploss*,{dagger}, Ingrid Leiner* and Eric G. Pamer2,*

* Infectious Diseases Service, Immunology Program, Department of Medicine, Sloan-Kettering Institute, Memorial Sloan-Kettering Cancer Center, New York, NY 10021; and {dagger} Immunology Program, Weill Graduate School of Medical Sciences of Cornell University, New York, NY 10021


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Prime-boost immunization 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 5–7 days following primary immunization. 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 populations.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Memory CD8 T cells are essential for immunity to many viral, bacterial, and protozoal pathogens (1). Upon primary activation by foreign Ag, CD8 T cells follow a program of proliferation and differentiation into CTL armed with effector functions that enable pathogen clearance or containment (2, 3, 4, 5, 6, 7, 8). After the expansion phase, the majority of Ag-specific CD8 T cells undergo programmed cell death, leaving a population of memory CD8 T cells that swiftly proliferate upon secondary antigenic challenge. Substantial evidence suggests that CD8 T cells transit through the effector phase before entering the memory pool (9, 10), but it remains unclear when, during the primary immune response, effector T cells differentiate into long-lived memory cells. A recent report showed that surface expression of the IL-7R {alpha}-chain (IL-7R{alpha}) distinguishes a small population of CD8 effector T cells at the peak of the primary response that gives rise to long-term memory cells (11); however, it remains unclear when, following priming, memory T cells acquire the ability to undergo extensive proliferation in response to Ag. Although it was previously demonstrated that increased CD8 T cell proliferation can be induced by additional Ag administration within 1 wk following primary bacterial infection (12), a more recent report analyzing the CD8 T cell response to lymphocytic choriomeningitis virus (LCMV)3 infection concluded that memory T cells do not proliferate upon re-encountering Ag for 3 wk following priming (13). LCMV infection, however, induces extraordinarily large primary immune responses (2, 3), and therefore may not reflect the type of T cell priming that occurs with vaccines.

To explore the kinetics of memory development and the factors that influence the magnitude of memory cell expansion in the setting of a more typical CD8 T cell response, we examined mice infected with the intracellular bacterial pathogen Listeria monocytogenes. Peak CD8 T cell responses to L. monocytogenes infection occur 8 days following bacterial inoculation; subsequently, T cells contract into stable memory populations within 14–21 days (14). In this study, we show that a subset of Ag-specific CD8 T cells can undergo recall proliferative responses upon secondary encounter with Ag within 5–7 days of primary infection, even before the primary T cell response begins to subside. Interestingly, we find that the rapid elimination of Ag by effector cells during an ongoing immune response prevents the activation and proliferation of memory cells, explaining the apparent lack of functional memory CD8 T cells at early time points after primary immunization. The ability of memory T cells to proliferate is revealed, however, when the boosting dose of Ag exceeds the clearance capacity of effector T cells. Remarkably, boosting at early or late time points following priming is similarly effective at enhancing both the size of secondary memory T cell populations and protective immunity to subsequent pathogen challenge. These findings have significant implications for the design and delivery of prime-boost vaccines.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice, bacteria, and viruses

BALB/cJ were obtained from The Jackson Laboratory (Bar Harbor, ME). BALB/c Thy-1.1-congenic mice were provided by C. Surh (Scripps Research Foundation, La Jolla, CA). Wild-type (WT) L. monocytogenes strain 10403S, the ActA–/– strain of L. monocytogenes (15) (both provided by D. Portnoy, University of California, Berkeley, CA), and the LLO92Ser mutant strain of L. monocytogenes (mutation of the tyrosine in position 92 of listeriolysin to a serine (16)) were grown in brain-heart infusion (BHI) broth (BD Biosciences, Sparks, MD). Vesicular stomatitis virus (VSV) expressing the L. monocytogenes listeriolysin O (LLO) and p60 epitopes was generated by cloning a PCR-amplified fragment of the L. monocytogenes p60 protein encompassing the p60217–225 and p60449–457 epitopes into pVSV-XN2 (kindly provided by J. Rose, Yale University, New Haven, CT) using its unique XhoI and NheI sites. A 64-bp linker encoding LLO91–99 was then ligated in frame with the p60 fragment at the XhoI site to generate a fusion LLO-p60 protein in pVSV-XN2. Recombinant VSV-LLO-p60 viruses were then obtained, as previously described (17). Briefly, the LLO-p60-containing plasmid was transfected along with pBS-N, pBS-P, and pBS-L helper plasmids into baby hamster kidney cells infected with recombinant vaccinia virus expressing T7 polymerase (vTF7-3). Supernatants were recovered 48 h later, spun down to remove debris, and filtered through a 0.2-µm filter to remove vTF7-3. The cleared supernatants were used to infect baby hamster kidney monolayers. Supernatants were recovered 48 h after the secondary infection, aliquoted, and stored at –80°C. Viral titers were determined by plaque assay.

Primary infections of mice were performed by injecting 2000 bacteria or 5 x 106 PFU of virus diluted in PBS into the tail vein. Viable bacterial counts in infected mice were determined by homogenizing spleens in PBS containing 0.1% Triton X-100 and plating on BHI agar plates.

Peptides, Abs, tetramers, and flow cytometry

Peptides were synthesized by Research Genetics (Huntsville, AL). The following Abs directed to mouse cell surface Ags were purchased from BD PharMingen (San Diego, CA): anti-CD8a PerCP (53-6.7), anti-Thy-1.2 allophycocyanin (53-2.1), and anti-CD62L allophycocyanin (MEL-14). Tetrameric H-2Kd/peptide complexes were generated, as described (12). Approximately 5 x 105 to 1 x 107 cells per sample were incubated on ice for 1 h with saturating concentrations of Abs and tetramers in FACS staining buffer (PBS, 1% FCS, 0.05% sodium azide). Labeled cells were washed with FACS buffer, fixed in PBS containing 2% paraformaldehyde, and analyzed on a BD-LSR flow cytometer (BD Biosciences) using CellQuest or FlowJo (Tree Star, San Carlos, CA) software.

CFSE labeling

Splenocytes depleted of RBC were washed in PBS and resuspended at 5 x 107/ml in PBS containing 10 µM CFSE (Molecular Probes, Eugene, OR). After incubating at 37°C for 10 min in the dark, cells were immediately washed with cold RPMI 1640 medium (Life Technologies, Grand Island, NY) with 10% FCS before resuspending in PBS for injecting i.v. into mice.

In vivo cytotoxicity assay

Analysis of in vivo cytotoxicity was conducted similarly to the published protocol (18, 19). BALB/c splenocytes were divided into two populations and labeled with CFSE at a concentration of 3 µM (CFSEhigh) or 0.3 µM (CFSElow). CFSEhigh cells were pulsed with 10–6 M LLO91–99 peptide for 1 h at 37°C in the dark, while CFSElow cells remained unpulsed. Subsequently, CFSEhigh cells were washed and mixed with equal numbers of CFSElow cells before injecting 1.5 x 107 total cells per mouse. Spleens from recipients were taken 15 h later for flow cytometric analysis to measure in vivo killing, as indicated by loss of the CFSEhigh Ag-pulsed population relative to the control CFSElow population.

Adoptive transfers

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 x 107 cells/recipient. Ampicillin (Sigma-Aldrich, St. Louis, MO) was given to donor mice at 2 mg/ml in their drinking water 5 days before sacrificing to ensure that no viable bacteria were transferred along with prepared splenocyte suspensions into naive recipients. Some recipients were infected 24 h later with 2000 WT L. monocytogenes, and expansion of donor cells, distinguished by Thy-1.2 expression, was assessed by flow cytometry at various times after infection.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Ag reintroduced during the primary response is rapidly cleared

To determine the kinetics of CD8 T cell memory generation during bacterial infection, we primed naive mice with a sublethal dose of 2000 WT L. monocytogenes and then rechallenged mice 7 or 90 days later with 104 bacteria. CD8 T cells specific for the immunodominant LLO91–99/H-2Kd epitope were quantified by MHC-peptide tetramer staining 5 days after the second bacterial inoculation. As expected, there was robust expansion of LLO91–99-specific CD8 T cells after rechallenge of mice immunized 90 days previously (Fig. 1a). In contrast, mice boosted 7 days after primary infection exhibited very little memory T cell expansion in comparison with nonboosted mice (Fig. 1a).



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  		FIGURE 1. In vivo CTL activity after primary immunization correlates with rapid clearance of reintroduced Ag and diminished secondary CD8 T cell expansion 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 bacteria. Spleens were harvested 5 days later, and expansion of LLO91–99/H-2Kd-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 showing similar results. b, In vivo CD8 T cell cytolytic activity peaks 7 days after primary infection and gradually wanes. Equal numbers of CFSEhigh, LLO91–99 peptide-pulsed BALB/c splenocytes and CFSElow, nonpulsed BALB/c splenocytes were coinjected i.v. into BALB/c 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 CFSE+ splenocytes in recipient mice. Numbers at the top of each plot represent the percentage of CFSElow or CFSEhigh cells of total CFSE+ donor cells recovered. Results are representative of two mice per group yielding similar data. c, Ag reintroduced early after primary immunization is rapidly cleared. After primary immunization, mice were challenged 7, 14, or 21 days later with 106 WT L. monocytogenes, and the number of viable 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.

 
Although these results are consistent with the notion that memory T cells require several weeks to develop following primary immunization (13), an alternative explanation is that the secondary bacterial challenge is cleared with different kinetics at early and late time points following primary infection. Indeed, recent studies demonstrated that swift activation of Ag-specific T cells following primary infection rapidly eliminates APCs, thereby restricting additional T cell priming (20, 21). To explore this possibility, we measured in vivo CTL activity using LLO91–99-coated target cells at intervals after primary L. monocytogenes infection. Ag-specific cytotoxicity could be observed within 2 days of infection, consistent with studies documenting the rapid development of CD8 T cell effector function (18, 19, 20, 22) (Fig. 1b). In vivo cytolytic activity peaked 7 days after bacterial inoculation, corresponding with the peak of CD8 T cell expansion, and subsequently waned to low, but detectable levels 14–21 days after infection. To determine whether in vivo cytolytic activity correlated with Ag clearance, we rechallenged mice with 106 bacteria 7, 14, or 21 days following primary infection and measured the number of viable L. monocytogenes in spleens 24 h later. Mice rechallenged 2–3 wk after primary infection had ~1000-fold more bacteria in spleens than mice rechallenged 7 days following priming (Fig. 1c), supporting the notion that large numbers of activated effector T cells promote rapid Ag clearance.

High dose Ag rechallenge within 1 wk of vaccination elicits secondary CD8 T cell expansion

To determine whether increasing the Ag dose at early time points following primary immunization could overwhelm the clearance capacity of the effector T cell response, and thereby uncover the recall proliferative potential of Ag-specific T cells, we primed mice with 2000 L. monocytogenes and rechallenged mice 7 or 90 days later with 2,000, 104, 105, or 106 bacteria. The expansion of LLO91–99-specific CD8 T cells was examined 5 days later. Although boosting with 2,000–10,000 bacteria 7 days after primary immunization did not induce significant expansion of Ag-specific CD8 T cells, mice immunized 90 days previously demonstrated significant secondary CD8 T cell expansion, even at the lowest rechallenge dose (Fig. 2a). Higher rechallenge doses (105–106 organisms), however, resulted in significant (>8x) secondary expansion of LLO91–99-specific CD8 T cells 7 days following priming (Fig. 2a). Indeed, this booster effect was also seen in mice rechallenged with high doses of L. monocytogenes as early as 5–6 days following primary infection (data not shown). These results indicate that the capacity of primed, Ag-specific T cells to undergo memory-like expansion in response to a second encounter with Ag is present at early time points during the primary CD8 T cell response.



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  		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, or 106 bacteria. Spleens were harvested 5 days later, and expansion of LLO91–99/H-2Kd-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 106 WT L. monocytogenes. Total numbers of LLO91–99-specific T cells from nonboosted ({square}) and boosted ({blacksquare}) mice were determined 5 days later by flow cytometry. Results are representative of two mice per group.

 
To determine whether the efficiency of in vivo Ag clearance inversely correlates with the extent of memory T cell expansion upon boosting, we rechallenged mice with 106 bacteria 7, 14, or 21 days following priming with L. monocytogenes. All mice fully cleared bacteria 5 days following rechallenge when secondary CD8 T cell expansion was measured, regardless of the time of boosting. Although substantial LLO91–99-specific T cell expansion was seen in all groups, incrementally enhanced proliferation was seen when rechallenge was delayed by 2 and 3 wk (Fig. 2b). Similar trends were noted for CD8 T cell responses to the Listeria p60217–225 and p60449–457 H-2Kd epitopes (data not shown). Thus, as the efficiency of in vivo Ag clearance diminishes, the magnitude of the memory T cell response to similar Ag doses increases.

Secondary effector responses inhibit memory CD8 T cell expansion

To determine whether rapid in vivo Ag clearance was also capable of inhibiting the expansion of long-term memory CD8 T cells, we primed mice with WT L. monocytogenes and boosted them 3 mo later with 0, 2000, 104, or 105 L. monocytogenes LLO92Ser, a strain that lacks the LLO91–99 epitope (16). Mice were then rechallenged 5 days later with 105 WT L. monocytogenes to stimulate expansion of LLO91–99-specific memory CD8 T cells. We predicted that mice boosted with L. monocytogenes LLO92Ser would generate secondary effectors specific for all bacterial epitopes except LLO91–99, and that this response would rapidly clear the subsequent rechallenge with WT L. monocytogenes, thereby preventing the activation of LLO91–99-specific memory CD8 T cells. Consistent with this prediction, immune mice that were not boosted with L. monocytogenes LLO92Ser exhibited massive secondary expansion of LLO91–99-specific CD8 T cells after rechallenge with WT L. monocytogenes (Fig. 3). In contrast, mice boosted with as little as 2000 L. monocytogenes LLO92Ser were unable to mount significant secondary LLO91–99-specific CD8 T cell responses upon WT L. monocytogenes rechallenge, supporting the notion that an ongoing Listeria-specific effector response can also prevent the activation of long-lived Listeria-specific memory CD8 T cells.



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  		FIGURE 3. Reduced memory expansion in the presence of secondary effectors. Mice immunized 95 days previously with 2000 WT L. monocytogenes were boosted with 0, 2000, 104, or 105 LLO92Ser L. monocytogenes and rechallenged 5 days later with 105 WT L. monocytogenes. Expansion of LLO91–99/H-2Kd-specific T cells was examined by flow cytometry another 5 days later. Density plots are gated on live CD8 T cells and are representative of two mice per group. The first plot on the left shows splenic LLO-specific CD8 T cells in nonboosted immune mice.

 
Efficient induction of early memory CD8 T cell responses by heterologous prime-boost vaccination

Recently described vaccination schemes use a heterologous prime-boost strategy in which primary immunization is performed using target Ags expressed in one vector and boosting is conducted using similar Ags expressed in a different vector, such as the consecutive use of DNA vaccines, followed by attenuated viral vectors expressing the same proteins (23, 24, 25, 26, 27, 28). This approach generates potent cellular immune responses compared with the use of a single vector and minimizes the effect of neutralizing Abs and T cell responses induced during priming. Because residual effector responses to primary immunization appeared to interfere with the expansion of memory T cells upon rechallenge, we postulated that early memory responses to LLO91–99 should be enhanced upon rechallenge with an infectious agent that expresses this epitope, but is otherwise antigenically distinct from L. monocytogenes. We therefore generated a strain of vesicular stomatitis virus that expresses the L. monocytogenes LLO91–99 and subdominant p60217–225 and p60449–457 epitopes (VSV-LLO-p60). After immunizing mice with this virus, we rechallenged animals with attenuated ActA-deficient L. monocytogenes 7, 14, 21, or 28 days later and measured expansion of LLO91–99-specific CD8 T cells. Using this heterologous prime-boost strategy, we detected nearly maximal memory responses 7 days after priming (Fig. 4), demonstrating not only that the capacity for memory expansion is present at this early time point following CD8 T cell priming, but also that boosting with a heterologous vector can efficiently overcome effector responses that limit the induction of memory responses.



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  		FIGURE 4. Heterologous prime-boost vaccination demonstrates that memory CD8 T cells are generated rapidly. BALB/c mice infected 7, 14, 21, or 28 days previously with 5 x 106 PFU VSV-LLO-p60 were challenged with 106 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 ({square}) and boosted ({blacksquare}) mice challenged at the indicated day after primary infection.

 
Early and late memory CD8 T cells exhibit similar expansion kinetics upon secondary encounter with Ag in adoptive transfer recipients

Secondary CD8 T cell expansion following early Ag re-exposure might reflect a large number of effector T cells undergoing a few additional rounds of proliferation or, alternatively, a smaller subset of the responding T cells undergoing extensive proliferation. To differentiate between these alternatives, we adoptively transferred CFSE-labeled splenocytes from naive mice or mice infected 8 vs 22 days previously with L. monocytogenes into naive Thy-1.1 congenic mice, and examined the expansion of transferred, Ag-specific T cells after infection of recipient mice. In the absence of infection, donor CD8 T cells from all groups of mice did not divide (Fig. 5a). Five days after bacterial infection, there was no detectable expansion of CD8 T cells from naive donors, but CD8 T cells from day 8 and 22 immune mice had extensively proliferated, as reflected by loss of CFSE, giving rise to remarkably similar populations of LLO91–99-specific CD8 T cells. Expansion of LLO91–99-specific T cells was Ag driven and not due to nonspecific inflammation, because infection of recipients with L. monocytogenes LLO92Ser stimulated secondary expansion of CD8 T cells from immune donors without generating LLO91–99-specific CD8 T cells.



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  		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 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 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 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 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 CD8+Thy-1.2+ 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 either left unboosted (open symbols) or challenged with 106 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.

 
To compare the kinetics of memory T cell expansion, we transferred CFSE-labeled immune splenocytes from day 8 or 46 infected mice into naive mice and measured donor CD8 T cell expansion at 2, 4, and 6 days following recipient infection. Pretransfer analysis of CD8 T cells from day 8 infected donors showed a substantial population of CD62Llow LLO91–99-specific effectors, while only a small number of LLO91–99-specific memory CD8 T cells, mostly CD62Lhigh, were found in day 46 infected donor mice (Fig. 5b). No detectable division of donor T cells was observed 2 days after infection of recipient mice, but by days 4 and 6, similar frequencies of expanded LLO91–99-specific CD8 T cells were detected among day 8 and 46 infected donor T cells (Fig. 5b). The mean CFSE fluorescence intensities among the expanded cells were similar, indicating that cells from both types of donors divided at similar rates. Plotting the absolute number of LLO91–99-specific donor CD8 T cells that expanded from day 8 and 46 infected mice over time demonstrated nearly identical expansion kinetics (Fig. 5c). Thus, despite the initial disparity in the number of Ag-specific CD8 T cells at the time of transfer, secondary expansion of donor CD8 T cells from recently or remotely immunized mice was similar, indicating that the T cell populations that underwent proliferation in response to Ag re-exposure were similar in size and responsiveness. This result is most readily explained by similarly small subsets of memory T cells in both groups undergoing extensive division. The similar extent of CFSE dilution in responding T cell populations demonstrates that they have undergone cell division a similar number of times. On the basis of CFSE intensity, we estimate that responding T cells in both experimental groups have undergone ~7 divisions. The similar 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 donors 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 stable secondary memory CD8 T cell populations, mice vaccinated with VSV-LLO-p60 either 7 or 28 days previously were challenged with ActA–/– L. monocytogenes, and the LLO91–99-specific CD8 T cell population was tracked over time. Mice boosted at day 7 or 28 postimmunization exhibited substantial memory responses 5 days later, with ~2-fold greater numbers of Ag-specific T cells in mice boosted at day 28 relative to day 7 boosted animals (Fig. 5d). The expanded memory populations contracted similarly to ~12–13% of the peak response after 1 mo, with Ag-specific CD8 T cells persisting at levels well above those of nonboosted animals. Thus, early and delayed boosting both generate significant numbers of long-lived, Ag-specific memory CD8 T cells.

Early and delayed boosting generate similar protective immunity

To determine whether early and delayed boosting induced similar levels of protective immunity, we immunized mice with 2000 WT L. monocytogenes, boosted animals 1 or 4 wk later with VSV-LLO-p60, and then challenged both groups of mice 1 mo later with 5 x 104, 105, or 5 x 105 WT L. monocytogenes. As controls, we also challenged naive mice and mice that were immunized 1 mo previously with 2000 WT L. monocytogenes, but not boosted with VSV-LLO-p60. As expected, naive mice exhibited progressively increasing numbers of bacteria in the spleen 24–72 h after infection, and those given the highest dose of L. monocytogenes died by day 3 (data not shown). In contrast, nonboosted, immune mice appeared healthy and had lower, albeit significant, numbers of bacteria 24 h after challenge with the three doses of L. monocytogenes, but these organisms were gradually cleared over the subsequent 2 days (Fig. 6). Remarkably, mice immunized with L. monocytogenes and boosted 7 or 28 days later with VSV-LLO-p60 both showed markedly increased protective immunity relative to nonboosted immune mice, as reflected by the sharply reduced numbers of bacteria that could be cultured from the spleen at all times following L. monocytogenes challenge. Although slightly enhanced protective immunity at 24 h postchallenge was observed after delayed vs early boosting with high doses of L. monocytogenes challenge, it was clear that both early and delayed boosting successfully conferred several logs of protection early after challenge relative to unboosted animals and allowed mice to clear a subsequent lethal L. monocytogenes challenge much more rapidly than nonboosted mice. Thus, the timing of the boost did not alter its ability to enhance the memory responses of challenged mice, providing strong support for the functionality of memory T cells expanded at early times after primary immunization.



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  		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 x 106 PFU VSV-LLO-p60. Mice were then challenged 30 days later with 5 x 104, 105, or 5 x 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.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Understanding when immunological memory develops is important for the rational design of vaccines. Our findings indicate that long-lived, Ag-specific memory CD8 T cells are already formed within 5–7 days of primary infection, and can be stimulated to undergo rapid and robust expansion upon early secondary encounter with Ag. This is observed in priming and boosting with the same pathogen and even more dramatically in heterologous prime boosting using viral and bacterial vectors. Importantly, early memory expansion does not reflect the stimulation of large numbers of effector T cells to undergo a few additional rounds of division, but rather reflects the extensive proliferation of a small subset of T cells embedded within the greater responding T cell population. Our results are consistent with recent data indicating that memory cell precursors can be found amid the primary response (11). However, we show that these cells already have the capacity to undergo secondary expansion upon early Ag re-encounter. Further evidence for these cells being bona fide memory T cells is provided by our data showing that these expanded cells provide robust long-term protective immunity, because both early and delayed boosting elicited similar levels of protection that were significantly higher than that of nonboosted mice when mice were challenged with the same pathogen 1 mo later.

We propose that, soon after infection, a subset of activated Ag-specific effector CD8 T cells is selected for the long-lived memory pool and rapidly acquires the potential to undergo extensive expansion upon secondary Ag encounter. Such diversification would be consistent with the recent finding that CD4 effector T cells responding to influenza infection exhibit broad heterogeneity in functional capacity (29). Our results contrast with the slower rate of memory T cell development recently reported using the LCMV system (13), perhaps due to the unusually large number of effector CD8 T cells generated during primary LCMV infection (2, 3), which would rapidly eliminate any booster Ag introduced at early times after immunization and prevent the efficient stimulation of the small number of functional memory cells that may already be present. Furthermore, increasing the priming dose of LCMV can lead to clonal CD8 T cell exhaustion (30), suggesting that the massive CTL response to this virus may not be representative of the type of CD8 T cell response elicited by most currently investigated vaccines.

Our finding that increased doses of Ag can induce secondary T cell expansion early after primary immunization suggests that in vivo Ag presentation may be extended by increased doses of Ag. This is an interesting possibility because we and others (20, 21) recently showed that in vivo functional Ag presentation to prime T cells actually occurs within only a brief period of ~2–3 days after infection, due to swiftly acquired CTL activity that eliminates professional APC. Higher Ag doses might lengthen this period of Ag presentation, but this may be unnecessary for T cell priming because we and other groups have shown that T cell responses are largely programmed within the first 24 h of Ag encounter, resulting in multiple rounds of Ag-independent proliferation over several days (5, 6, 7, 8). However, it is tempting to speculate that extended Ag presentation may allow the recruitment of additional T cell clonotypes into the response that may not normally be primed once CTL effector activities have down-regulated in vivo Ag presentation.

Our study provides a novel explanation for the observed kinetics of memory T cell development whereby CTL-mediated feedback inhibition during an ongoing immune response can prevent newly formed, functional memory T cells from being activated early after initial immunization. We have shown in this work that, with sufficient antigenic stimulation to overcome rapid Ag clearance by the effector activities present during the primary response, secondary responses that provide increased immunological protection can be elicited even before the primary response has declined. This clearly has important implications for the logistics of vaccine delivery in clinical settings because vaccination regimens may be completed under much shorter time periods. Indeed, the rapid development of memory T cells suggests that vaccines containing an early and delayed release component, such as a slow degradable capsule containing a second, larger dose of Ag to be administered with the initial inoculating dose of Ag, may enable prime boosting with a single visit to the clinic.


   Acknowledgments
 
We thank Rielle Giannino, An Tran, and Ewa Menet for excellent technical support. The vTF7-3 was obtained through the National Institutes of Health AIDS Research and Reference Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Diseases, National Institutes of Health (contributed by Drs. Tom Fuerst and Bernard Moss).


   Footnotes
 
1 This work was supported by National Institutes of Health Grants AI-39031 and AI-42135, an Arthritis Foundation postdoctoral fellowship (to P.W.), and a Cancer Research Institute predoctoral fellowship (to A.P.). M.L.-T. is a Rosenwald Family/DeVaan-Irvington Institute postdoctoral fellow. Back

2 Address correspondence and reprint requests to Dr. Eric G. Pamer, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021. E-mail address: pamere{at}mskcc.org Back

3 Abbreviations used in this paper: LCMV, lymphocytic choriomeningitis virus; BHI, brain-heart infusion; LLO, listeriolysin O; VSV, vesicular stomatitis virus; WT, wild type. Back

Received for publication December 5, 2003. Accepted for publication March 25, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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