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The Journal of Immunology, 2008, 180, 1131-1138
Copyright © 2008 by The American Association of Immunologists, Inc.
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CD8 T Cell Expansion and Memory Differentiation Are Facilitated by Simultaneous and Sustained Exposure to Antigenic and Inflammatory Milieu1

Angela Shaulov* and Kaja Murali-Krishna2,*,{dagger}

* Department of Immunology and {dagger} Washington National Primate Center, University of Washington School of Medicine, Seattle, WA 98195


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Understanding the factors contributing to the generation of immune memory is important for rational vaccine design. In this study, we addressed the individual and combined roles of Ag and inflammation in sustaining the ability of primed CD8 T cells to clonally expand and differentiate into memory cells. We transferred CD8 T cells that were primed for a brief period into naive mice, mice infected with a pathogen not carrying the specific Ag (inflammation only), mice infected with a pathogen carrying the donor cell-specific Ag (inflammation plus Ag), or into mice exposed to soluble Ag (Ag only). We found that the donor CD8 T cells continued to proliferate in all the four conditions, but their ability to clonally expand and differentiate into memory cells was ~1000-fold higher when transferred into mice acutely infected with pathogen carrying the relevant Ag. Memory cells generated under conditions of sustained exposure to inflammation and Ag during the priming phase were superior in their ability to elicit recall responses on a per cell basis. Thus, simultaneous and sustained exposure of donor CD8 T cells to inflammatory and antigenic stimuli, following the initial priming phase, leads to the greatest expansion of CD8 T cells at the peak of the immune response and induces an optimal memory differentiation program. These results suggest that vaccination strategies should attempt to provide sustained exposure to Ag plus inflammation but not either alone following the initial priming.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The generation of immunological memory is the end result of a productive, adaptive immune response and is the ultimate goal of vaccination (1, 2). A successful vaccine needs to induce a large number of memory cells equipped with a heightened ability to respond to a recall challenge. Many vaccination strategies attempt to induce "more and most fit" memory CD8 T cells because of their importance in controlling a broad range of viral and intracellular bacterial infections as well as malignancies. Therefore, an understanding of the factors that contribute to optimal CD8 T cell memory generation is critical for devising improved vaccine and therapeutic strategies.

Live replicating agents generally induce more potent primary and memory CD8 T cell responses than inactivated vaccines. The precise mechanisms affecting the quantity and quality of memory T cells, following the initial priming by a live agent, is less clear. Several recent studies have indicated that events during the initial priming stage of the infection, including Ag availability, inflammatory stimuli, costimulatory molecules, CD4 T cell help, and secreted growth factors, have long lasting programming effects on the quantity and quality of the memory cells (3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17).

A brief antigenic encounter is sufficient to program CD8 T cell proliferation (5), but the expansion and the ability to form memory (determined by the survival of the proliferating cells) is dependent upon several cell intrinsic and extrinsic factors (11, 12, 13, 14, 15, 16), the nature of which is not completely understood.

The inflammation induced by live infection can greatly enhance the survival of the clonally proliferating CD8 T cells during an acute phase of infection (11, 12, 13). Paradoxically, it has been observed that inflammation can also increase T cell contraction at later phases, thereby decreasing the number of memory cells under the conditions of limiting antigenic stimulus (7, 14). However, the individual influence of Ag and inflammation on the ability of primed CD8 T cells to expand and differentiate into memory is less clear.

To address the individual and combined influence of Ag and inflammation on primed CD8 T cells, we stimulated OVA-specific OT-1 CD8 T cells, in vivo or in vitro, and transferred them into naive mice (in which neither inflammation nor Ag were available), into mice infected with pathogen not carrying the OVA epitope (inflammation only), into mice infected with pathogen carrying the OVA epitope (inflammation plus Ag), or into mice injected with soluble OVA protein (Ag only). Consistent with previous reports (5, 6), we found that primed CD8 T cells proliferated in a programmed manner under all four conditions. However, primed OT-1 CD8 T cells exposed to both inflammation and Ag generated higher (~1000-fold more) numbers of effector and memory cells than did the CD8 T cells that received inflammatory signals only, antigenic signals only, or neither. The memory CD8 T cells generated under conditions of continued exposure to inflammation and Ag were superior in their ability to elicit recall responses than the memory CD8 T cells generated under conditions of continued exposure to inflammation only, Ag only, or neither. Thus, continued exposure of the primed CD8 T cells to Ag and inflammation, but neither alone, greatly facilitates generation of an optimal memory CD8 T cell competent of eliciting better recall responses. These results open novel avenues for further understanding of the mechanisms contributing to the generation of optimal immunological memory and have potential implications for vaccine development strategies.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

Wild-type (WT)3male C57BL/6 (B6 CD45.2) and B6.SJL-PtprcaPep3b/BoyJ (B6 CD45.1) mice were obtained from Jackson ImmunoResearch Laboratories and used at 5–6 wk of age. OT-1 mice expressing the OVA257–264/Kb-specific TCR and congenic for CD45.1 and CD45.2 markers were bred in house. All mice were kept under specific pathogen-free conditions in compliance with the guidelines of the Institutional Animal Use and Care Committee.

Bacteria and viruses

Listeria monocytogenes that expresses a secreted form of OVA (rLm-OVA) (18) and WT Listeria monocytogenes (Lm WT) were grown as described (9). For primary infections, mice were injected with 2 x 103 CFU of Lm WT i.p. and 10 mg of OVA protein in PBS i.p. or with 4 x 103 CFU of rLm-OVA i.p. Lymphocytic choriomeningitis virus (LCMV) Armstrong strain was plaque purified and grown in BHK cells and titered on Vero cells. Mice were injected with 2 x 105 PFU of LCMV i.p. Recombinant vesicular stomatitis virus-expressing OVA (rVSV-OVA) was previously described (19). For rechallenge infections, mice were injected with 2 x 106 PFU of rVSV-OVA i.v. and sacrificed 4 days later.

Dendritic cell (DC) generation and activation

Dendritic cells were generated from bone marrow of WT B6 CD45.2 mice using recombinant murine GM-CSF as previously described (20). At day 6 of culture, DCs were stimulated with 10 µg/ml poly(I:C) and 1 µg/ml murine anti-CD40 mAb overnight. Subsequently, the DCs were either loaded with 0.1 µg/ml SIINFEKL peptide for 5 h or left untreated.

CD8 T cell purification, priming, and adoptive transfers

OT-1 TCR transgenic (Tg) CD8 T cells were sorted from spleens of naive mice using the Miltenyi Biotec CD8 T cell isolation kit. Untouched purified CD8 T cells were >95% pure as assessed by flow cytometry. For in vitro priming, purified OT-1 CD8 T cells were cocultured with SIINFEKL (OVA257–264) peptide-loaded GM-CSF bone marrow-derived DCs for 48 h at 37°C and 5% CO2. Where applicable, Brefeldin A was added in the last 5 h of culture to measure IFN-{gamma} production. Following in vitro stimulation, CD8 T cells were removed from culture and washed extensively in serum-free media; 2.5 x 105 OT-1 CD8 T cells were adoptively transferred into naive recipients. Recipients were left untreated or infected 6–12 h later with 2 x 105 PFU of LCMV i.p. (inflammation only group), 2 x 105 PFU of LCMV and 10 mg OVA protein in PBS (both i.p.) (inflammation and Ag group), or 10 mg OVA only i.p. (Ag only group). Before adoptive transfer, the phenotype of CD8 T cells was assessed by flow cytometry, by staining with anti-CD62L, anti-CD69, and anti-CD25 Abs. For in vivo priming, 3 x 106 purified OT-1 CD8 T cells were adoptively transferred i.v. into naive recipients. Twenty four hours later, mice were infected with 2 x 103 CFU of Lm WT and 10 mg of OVA protein in PBS (both i.p.). Forty eight hours post infection, CD8 T cells from spleens and lymph nodes were isolated using the Miltenyi Biotec CD8 T cell isolation kit. Subsequently, 1.4 x 104, 8 x 104, or 2 x 105 OT-1 CD8 T cells were adoptively transferred i.v. into naive hosts and infected with 2 x 103 CFU of Lm WT i.p. (inflammation only group), 4 x 103 CFU of Lm-OVA i.p. (inflammation and Ag group) 4–6 h later (unless stated otherwise), or left untreated (neither group). Before adoptive transfer, the phenotype and function of CD8 T cells was assessed by flow cytometry, by staining with anti-CD62L, anti-CD69, anti-CD25, anti-CD43, anti-CD44, and anti-CD127 Abs and by measuring IFN-{gamma} production following SIINFEKL peptide restimulation in vitro.

Cell staining and flow cytometry

Single-cell suspensions were prepared from the spleen and lymph nodes at indicated time points. Peripheral blood samples were treated with ammonium chloride solution to lyse RBC. All Abs were purchased from BD Biosciences or eBioscience. For cell surface staining, cells were stained for 30 min on ice in FACS buffer (1% BSA and 0.01% NaN3 in PBS), washed twice, and fixed in 2% paraformaldehyde. For intracellular cytokine staining, splenocytes were cultured for 5 h in a 5% CO2 incubator at 37°C in the presence of 1 µg/ml brefeldin A and 0.1 µg/ml SIINFEKL (OVA257–264) peptide. Cell surface staining was performed as described above; intracellular cytokine staining was performed using a Cytofix/Cytoperm kit (BD Biosciences) according to the manufacturer’s instructions. BrdU staining was done using a BrdU flow kit (BD Biosciences) according to the manufacturer’s instructions.

CFSE labeling and in vivo BrdU pulsing

For CFSE labeling, cells were washed two times with serum-free medium, followed by incubation at room temperature with 5 µM CFSE for 7 min, quenching with 20% FCS, and washing with RPMI 1640 with 10% FCS. For BrdU pulsing, mice were injected with 1.5 mg BrdU i.p. 16 h before analysis.

Statistical analysis

Values of p were calculated using Student’s t test (two-tail distribution, equal variance) with p < 0.05 taken as statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
We have taken two different approaches to examine the effect of sustained exposure to Ag and inflammation on primed CD8 T cell expansion. In the first approach, we primed naive CD8 T cells in vitro for 48 h, transferred them into mice, and provided inflammation and Ag, inflammation only, Ag only, or neither.

To achieve in vitro priming, we generated bone marrow-derived(I:C) DCs using GM-CSF, as described previously (20). Subsequently, DCs were stimulated with poly and anti-CD40 mAb overnight. The poly(I:C) and anti-CD40 mAb treatment resulted in the up-regulation of CD86, CD40, and MHC II and secretion of MCP-1, TNF-{alpha}, and IL-6 (data not shown). Next, we ensured that under these conditions, DCs were able to prime naive CD8 T cells in vitro. To this end, GM-CSF bone marrow-derived DCs were stimulated overnight with poly(I:C) and anti-CD40, pulsed with SIINFEKL peptide, washed, and used to stimulate naive OT-1 TCR Tg CD8 T cells in vitro for 48 h. Stimulation with peptide-pulsed, but not unpulsed, DCs resulted in the up-regulation of CD69 and CD25 and down-regulation of CD62L (Fig. 1A). A fraction of activated OT-1 CD8 T cells were producing IFN-{gamma} in the last 5 h of 48 h stimulation (Fig. 1B).


Figure 1
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  		FIGURE 1. Simultaneous and sustained exposure to antigenic and inflammatory stimuli results in greater expansion of CD8 T cells on day 7. Bone marrow GM-CSF-derived DCs were stimulated with poly(I:C) and anti-CD40 mAb overnight and then left unloaded or loaded with SIINFEKL peptide for 5 h. Naive TCR Tg OT-1 CD8 T cells were sorted from spleen and cocultured for 48 h with bone marrow-derived DCs. A, Activation status of OT-1 CD8 T cells 48 h after in vitro stimulation with SIINFEKL peptide. Naive TCR Tg OT-1 CD8 T cells obtained from fresh splenocytes were used as a control. All histograms are gated on OT-1 CD8 T cells. The data are representative of three independent experiments. B, Same as A. In the last 5 h of culture, Brefeldin A was added to measure IFN-{gamma}. C, Naive TCR Tg CD45.1+ OT-1 CD8 T cells were sorted from spleen and cocultured for 48 h with bone marrow-derived, SIINFEKL-pulsed DCs. Subsequently, OT-1 CD8 T cells were removed from the culture, washed three times, and 250,000 were transferred i.v. into naive B6 CD45.2 recipients. Six to twelve hours later the animals were immunized with 2 x 105 PFU LCMV i.p. (inflammation only group); 2 x 105 PFU LCMV and 10 mg OVA both i.p. (inflammation and Ag group); 10 mg OVA i.p. (Ag only group); or left untreated. On day 7 post infection, the animals were sacrificed, and spleens were collected for analysis. Top, One representative animal of three is shown. All plots are gated on total lymphocytes. Percentage in italics represents the percent of OT-1 CD8 T cells among all CD8 T cells. Bottom, An average for three animals per group is plotted with SEM. Absolute number of OT-1 CD8 T cells per spleen is calculated based on the percentage of OT-1 CD8 T cells among all lymphocytes in spleen. The data are representative of three independent experiments. D, Same as for C. On day 5 post infection, the animals were sacrificed, and spleens were collected for analysis. Top, BrdU staining following 16 h BrdU pulse (1.5 mg i.p.). The percentages represent a fraction of cells BrdU+ in a given population. All dot plots are gated on CD8 T cells. Bottom, An average for three animals per group is plotted with SEM. The data are representative of two independent experiments. E, Same as for D. Top, Intracellular cytokine staining following 5 h of SIINFEKL stimulation in vitro. All dot plots are gated on OT-1 CD8 T cells. Bottom, An average of three animals per group is plotted with SEM. The data are representative of two independent experiments.

 
Using this system, we primed OT-1 CD8 T cells in vitro for 48 h and then transferred them into separate groups of mice (as depicted in Fig. 1C). The first group of mice was left untreated (neither Ag nor inflammation), the second was infected with LCMV (inflammation only), the third was primed with OVA along with LCMV infection (Ag plus inflammation), and the last group of mice was immunized with soluble OVA protein (Ag only). The recipient mice were analyzed at day 7 post infection. The results (Fig. 1C) show the following: First, in the absence of continued antigenic and inflammatory stimuli, the primed donor OT-1 CD8 T cells expanded marginally (<1 log). Second, sustained exposure to inflammation only (in the absence of Ag) did not significantly enhance their expansion. Third, sustained exposure to Ag only led to a slight (~1 log) increase in expansion. Lastly, simultaneous exposure to inflammatory milieu and Ag led to the greatest (>3 logs) expansion of CD8 T cells. Taken together, our results suggest the following: First, CD8 T cells primed for a short period in vitro can undergo programmed expansion when transferred into naive mice, but the extent of expansion is limited. Second, sustained exposure to Ag and inflammation following initial priming, but neither alone, can greatly facilitate this expansion.

Reduced CD8 T cell expansion in the absence of Ag and inflammation was not simply due to an inability to proliferate, because a sizable fraction of the cells incorporated BrdU between days 5 and 5 1/2 post infection. Specifically, the ability of donor CD8 T cells in the LCMV only group to incorporate BrdU was similar to that of the LCMV and OVA group (Fig. 1D) despite a substantial difference in clonal expansion. However, BrdU incorporation was marginally lower in CD8 T cells from uninfected and OVA-only immunized groups (Fig. 1D). This suggests that primed CD8 T cells are programmed to proliferate in all four conditions, but their proliferation and accumulation was greatest with sustained exposure to Ag and inflammation following the initial 48 h priming. Therefore, extended exposure to an inflammatory and antigenic stimulation after the initial 48 h priming phase positively augments both proliferation and survival of the CD8 T cells.

Next, we examined the ability of OT-1 CD8 T cells derived from the different groups to produce IFN-{gamma}. Fig. 1E shows that upon SIINFEKL peptide restimulation in vitro, OT-1 CD8 T cells derived from all four conditions are able to produce IFN-{gamma}. The ability of OT-1 CD8 T cells derived from the LCMV-only group to produce IFN-{gamma} was not statistically different from that of OT-1 CD8 T cells derived from LCMV and OVA group. However, the OT-1 CD8 T cells derived from the OVA-only group produced less IFN-{gamma} than OT-1 CD8 T cells derived from the other three groups. Therefore, 48 h in vitro primed OT-1 CD8 T cells continue to retain the ability to produce IFN-{gamma} whether or not OT-1 CD8 T cells were further subjected to Ag, inflammation, both, or neither. However, sustained exposure to Ag alone partially diminished the ability to produce IFN- {gamma} (Fig. 1E). Thus, simultaneous exposure to inflammatory milieu and sustained antigenic stimulus facilitates the expansion, proliferation, and cytokine production by CD8 T cells during a peak of the immune response.

In the second approach, we primed CD8 T cells in vivo for 48 h and then transferred them into groups of mice. In vivo priming was achieved by transferring naive OT-1 CD8 T cells into congenically marked mice, followed by immunization with Lm WT and OVA. We ensured that OT-1 CD8 T cells had undergone Ag-specific activation by measuring CD25, CD69, CD43, and CD44 up-regulation and CD62L and CD127 down-regulation in the spleen and lymph nodes 48 h post immunization (Fig. 2A and data not shown). A substantial portion of in vivo activated OT-1 CD8 T cells produced IFN-{gamma} following in vitro peptide restimuation (Fig. 2B). At 48 h post immunization, we purified OT-1 CD8 T cells from these mice and transferred them into naive secondary hosts, which were either left untreated, infected with Lm WT, or infected with rLm-OVA 1 h post cell transfer as depicted in Fig. 2C. Donor OT-1 CD8 T cells were below the level of detection in the uninfected and Lm WT infected recipients on day 6 post cell transfer but were easily visible in mice infected with rLm-OVA (Fig. 2C). This result suggested that both in vitro and in vivo primed OT-1 CD8 T cells require simultaneous exposure to inflammatory milieu and sustained antigenic stimulus for potent expansion. The lack of detectable expansion of OT-1 CD8 T cells in Lm WT infected mice was not simply due to transient depravation of chemokines, cytokines, and other growth factors between the time of cell transfer and immunization, since a similar pattern was observed when, in the same experiment, we transferred 48 h in vivo primed OT-1 CD8 T cells into mice that were infected with Lm WT 2 days before cell transfer (Fig. 2D). OT-1 CD8 T cell expansion under condition of inflammation and Ag, however, was slightly (4- to 5-fold) higher in recipients infected before cell transfer than in recipients infected at the time of cell transfer. This may, perhaps, reflect the ability of OT-1 CD8 T cells to avail Ag soon after cell transfer in preinfected recipients.


Figure 2
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  		FIGURE 2. Simultaneous and sustained exposure to antigenic and inflammatory stimuli following the initial 48 h priming in vivo results in greater expansion of CD8 T cells. A, Naive TCR Tg CD45.1+ OT-1 CD8 T cells were sorted from spleen and adoptively transferred into B6 CD45.2 recipients. Twenty four hours later, the animals were immunized with 2 x 103 CFU Lm and 10 mg OVA (both i.p.). Activation status of OT-1 CD8 T cells 48 h post infection with Lm and OVA in spleen. The data are representative of three independent experiments. B, Same as for A. Intracellular cytokine staining following 5 h of SIINFEKL stimulation in vitro. C, Same as for A. Forty eight hours post infection, the animals were sacrificed, and OT-1 CD8 T cells were sorted with Mitenyi Biotec CD8 T cell kit by negative depletion. Fourteen thousand in vivo primed OT-1 CD8 T cells were transferred i.v. into naive B6 CD45.2 recipients. One hour later, the animals were immunized with 2 x 103 CFU Lm WT i.p. (n = 3), 4 x 103 CFU rLm-OVA i.p. (n = 3), or left untreated (n = 3). On day 6 post cell transfer, the animals were sacrificed, and spleens were collected for analysis. One representative animal of three is shown. Plots are gated on total lymphocytes. Percentage in italics represents the percent of OT-1 CD8 T cells among all CD8 T cells. D, Same as for C. Fourteen thousand in vivo OT-1 CD8 T cells were transferred i.v. into B6 CD45.2 recipients infected with indicated pathogen 2 days before cell transfer. On day 6 post cell transfer, the animals were sacrificed, and spleens were collected for analysis. One representative animal of three is shown. Plots are gated on total lymphocytes. Percentage in italics represents the percent of OT-1 CD8 T cells among all CD8 T cells.

 
To achieve better visualization of OT-1 CD8 T cells in Lm WT and uninfected groups, in the subsequent experiment, we purified 48 h in vivo primed OT-1 CD8 T cells and transferred a higher number (80,000) of cells into naive secondary hosts, which were either left untreated, infected with Lm WT, or infected with rLm-OVA. The animals were bled on days 5, 8, 15, and 30 post infection to monitor OT-1 CD8 T cell expansion, contraction, and memory formation. First, we observed that the primed OT-1 CD8 T cells expanded dramatically between days 5 and 8 under conditions of sustained exposure to Ag and inflammation. These cells contracted from day 15 onward and formed a stable memory population by day 30 (Fig. 3A; diamonds). Second, under conditions of sustained exposure to inflammation only, the primed OT-1 CD8 T cells did not expand as much as they did under the condition of inflammation and Ag, contracted below the limit of detection by day 8 and remained undetectable at day 30 (Fig. 3A; circles). The primed donor OT-1 CD8 T cells behaved similarly under conditions of no Ag and inflammation (Fig. 3A; triangles). These results confirmed that sustained exposure to Ag and inflammation following initial priming, but neither alone, resulted in the greatest expansion of the primed CD8 T cells and the ability to form memory.


Figure 3
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  		FIGURE 3. Simultaneous and sustained exposure to antigenic and inflammatory stimuli results in greater expansion of CD8 T cells at the peak of the immune response and stronger rechallenge to a heterologous pathogen. A, Naive TCR Tg CD45.1+ OT-1 CD8 T cells were sorted from spleen and adoptively transferred into B6 CD45.2 recipients. Twenty four hours later, the animals were immunized with 2 x 103 CFU Lm and 10 mg OVA (both i.p.). Forty eight hours post infection, the animals were sacrificed, and CD8 T cells were sorted with Mitenyi Biotec CD8 T cell kit by negative depletion. Eighty thousand OT-1 CD8 T cells were transferred i.v. into naive B6 CD45.2 recipients. Four to six hours later, the animals were immunized with 2 x 103 CFU Lm WT i.p. (n = 6), 4 x 103 CFU rLm-OVA i.p. (n = 6), or left untreated (n = 3). The animals were bled at days 5, 8, 15, and 30 post infection. An average percentage of OT-1 CD8 T cells among all CD8 T cells in the peripheral blood is plotted with SEM. Dot plots: Recovery of OT-1 CD8 T cells in the spleen on day 36 post infection. B, The animals were challenged with 2 x 106 PFU (i.v.) rVSV-OVA on day 36 post infection. Four days post challenge, the animals were sacrificed and OT-1 CD8 T cell frequency was determined by flow cytometry in spleen and peripheral blood. One representative animal of three is shown. All plots are gated on total lymphocytes. Percentage in italics represents the percent of OT-1 CD8 T cells among all CD8 T cells.

 
We next examined memory recall responses of OT-1 CD8 T cells in each of these groups of mice. We rechallenged each of the groups of mice with rVSV-OVA. OT-1 CD8 T cells became detectable in all three groups following rVSV-OVA challenge (Fig. 3B). This result suggested that there were some donor memory OT-1 CD8 T cells that were capable of eliciting recall responses even in naive and inflammation only groups, which were below the level of detection before rechallenge. This result also suggests that OT-1 CD8 T cells that were exposed to sustained antigenic and inflammatory stimuli following initial priming exhibited the most dramatic recall response following rVSV-OVA challenge.

We set out to determine whether those differences in expansion following rechallenge are due to intrinsic differences in OT-1 CD8 T cells generated under various priming conditions and reflect their ability to expand upon rechallenge on a per cell basis. To address this question, we performed the experiment outlined in Fig. 4A by transferring a much higher number (2 x 105) of in vivo primed OT-1 CD8 T cells into different groups. This facilitated our ability to track the memory cells compared with the experiment in Fig. 3A. In Fig. 4, A and B, we show that the primed OT-1 CD8 T cells were detectable in all groups. The expansion and contraction of OT-1 CD8 T cells could be detected in the uninfected group (neither Ag nor inflammation), Lm WT infected group (inflammation only), as well as in rLm-OVA infected group (Ag and inflammation). The expansion as well as the size of memory pool following contraction was greater when the OT-1 CD8 T cells were exposed to Ag and inflammation (Fig. 4B). The size of the memory pool was substantially lower when the primed OT-1 CD8 T cells were exposed to neither Ag nor inflammation or exposed to inflammation only (Fig. 4B). Additionally, we found that the percentage of OT-1 CD8 T cells in peripheral blood on day 123 was closely mirrored by the percentage of OT-1 CD8 T cells in the spleen on day 130. This system, while confirming the observations in Fig. 3A, also allowed us to test the ability of the memory cells generated under these different conditions to respond to rechallenge on a per cell basis.


Figure 4
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  		FIGURE 4. Simultaneous and sustained exposure to antigenic and inflammatory stimuli during the priming phase of the immune response leads to memory CD8 T cells that generate stronger recall responses to a heterologous pathogen. A, Naive TCR Tg CD45.2+ OT-1 CD8 T cells were sorted from spleen and adoptively transferred into B6 CD45.1 recipients. Twenty four hours later, the animals were immunized with 2 x 103 CFU Lm and 10 mg OVA (both i.p.). Forty eight hours post infection, the animals were sacrificed and CD8 T cells were sorted with Miltenyi Biotec CD8 T cell kit by negative depletion. Two hundred thousand OT-1 CD8 T cells were transferred i.v. into naive B6 CD45.1 recipients. Four to six hours later, the animals were immunized with 2 x 103 CFU Lm WT i.p. (n = 10), 4 x 103 CFU rLm-OVA i.p. (n = 9), or left untreated (n = 8). The animals were bled at days 8, 45, 75, and 123 post infection and sacrificed on day 130 post infection. One representative animal per group is shown. Data for the same animal in each group is shown from day 8 to 130. All plots are gated on total lymphocytes. Percentage in italics represents the percent of OT-1 CD8 T cells among all CD8 T cells. B, An average percentage of OT-1 CD8 T cells among all CD8 T cells in the peripheral blood is plotted with SEM. Note: Days 8–123—PBMC and Day 130—spleen. C, Same as for A. On day 130 post infection, the animals were sacrificed, and CD8 T cells were sorted with Miltenyi Biotec CD8 T cell kit by negative depletion. Five thousand OT-1 CD8 T cells were transferred i.v. into naive B6 CD45.1 recipients. Twenty four hours post cell transfer, the animals were infected with 2 x 106 PFU (i.v.) rVSV-OVA. Four days post challenge, the animals were sacrificed and OT-1 CD8 T cells frequency was determined by flow cytometry in spleen and lymph node. One representative animal of five is shown. Top two rows, Plots are gated on total lymphocytes. Percentage in italics represents the percent of OT-1 CD8 T cells among all CD8 T cells. Bottom two rows, Intracellular cytokine staining following 5 h of SIINFEKL stimulation in vitro. Percentage in italic represents the percent of OT-1 CD8 T cells producing IFN-{gamma}. D, An average for five animals per group is plotted with SEM. Absolute number of OT-1 CD8 T cells per spleen is calculated based on the percentage of OT-1 CD8 T cells among all lymphocytes in spleen.

 
To this end, we transferred equal numbers (5000) of sorted day 130 memory OT-1 CD8 T cells, generated in each of the groups, into new recipients. Fig. 4C shows that even though memory CD8 T cells from Lm WT (inflammation only) and uninfected groups (neither Ag nor inflammation) were able to generate recall responses, the extent of expansion 4 days after rechallenge with rVSV-OVA was significantly less compared with rLm-OVA group (inflammation and Ag) (Fig. 4D). The differences in the expansion did not correlate with the ability to produce IFN-{gamma} and TNF-{alpha}, as a similar fraction of OT-1 CD8 T cells from all three groups produced these cytokines (Fig. 4C and data not shown). Therefore, simultaneous and sustained exposure to inflammatory milieu and antigenic stimulus during the priming phase led to maximal expansion of CD8 T cells at the peak of the immune response and programmed generation of higher numbers of memory cells that are equipped with the ability to elicit better recall responses to a heterologous pathogen on a per cell basis.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
A deeper understanding of mechanisms contributing to programming and differentiation of CD8 T cells is valuable in the development of effective vaccines and adoptive immunotherapies for treatment of infectious disease and cancer. In this report, we examined the combined and individual roles of sustained exposure to Ag and inflammation following initial priming of the CD8 T cells. We show, using two different experimental models with inflammation induced by two distinct pathogens (L. monocytogenes & LCMV), that sustained and simultaneous exposure to antigenic and inflammatory stimuli led to the greatest expansion of previously activated CD8 T cells at the peak of the primary immune response, IFN-{gamma} production, and programmed proliferation. Withdrawal of either inflammation or Ag or both after the initial 48 h priming phase marginally impaired proliferation and IFN-{gamma} production but drastically limited clonal expansion, suggesting that optimal survival of the clonally proliferating CD8 T cells requires simultaneous exposure to both Ag and inflammation. The memory cells that were generated by providing sustained antigenic and inflammatory stimuli during priming were most efficient in eliciting recall responses on a per cell basis, suggesting that the memory differentiation programs are greatly facilitated by simultaneous and sustained exposure to inflammation and Ag following the initial 48 h priming.

The memory CD8 T cells that were formed under the conditions of an acute antigenic withdrawal or withdrawal of both stimuli expanded less following rechallenge but retained the ability to make effector cytokines. This suggests that the longer exposure to Ag and inflammation following initial priming may play a selective role in programming survival and expansion following rechallenge rather than the ability to acquire cytokine effector functions.

Our results are consistent with and add new insight to the concept of programming in CD8 T cell responses (3, 4, 5, 6, 8). Previous studies examined the role of shortened antigenic exposure on CD8 T cell programming and memory generation (3, 4, 9, 10), and found that a 2 h stimulus is sufficient to induce a program of proliferation. Priming for 24–48 h is needed to induce a program of expansion in vivo. But whether sustained exposure to Ag or inflammation beyond the initial priming period affects further expansion and memory differentiation programs remained unclear. In the present study, we found that longer exposure to Ag and inflammation greatly facilitated clonal expansion and memory differentiation programs. More importantly, our results show that both Ag and inflammation should be available simultaneously; neither of them alone is efficient in promoting initial clonal expansion, memory pool size, or programming the ability to elicit recall responses.

Previous studies, in which L. monocytogenes replication was shortened by antibiotic treatment, led to an understanding that 48 h priming in vivo is sufficient for inducing an expansion program that leads to maximal clonal expansion of CD8 T cells (3, 9). Our results, while reinforcing the concept of programming, show that the first 48 h stimulus is insufficient to induce a full-blown program of CD8 T cell expansion and memory generation. Although withdrawing either Ag or inflammation or both allowed continued proliferation, it greatly diminished clonal expansion, the size of the memory pool, and the ability of the memory cells to elicit a recall response. Our results are consistent with a recent study by Prlic et al. (10) showing that terminating Ag stimulation by targeted depletion of Ag presenting DCs using diphtheria toxin at 54 h (48 h post priming plus 6 h to eliminate Ag presenting DCs) leads to a better expansion of CD8 T cells compared with terminating Ag presentation at 24 h or earlier in vivo.

Why did previous studies lead to an understanding that the 48 h priming is sufficient for a full-blown program of CD8 T cell expansion? These reports clearly show that L. monocytogenes replication is completely inhibited by 48 h post infection (3, 9). However, this does not insure that all Ag is gone. Residual Ag can be sequestered in remote locations and nonlymphoid tissues to which the already primed effector CD8 T cells can selectively gain access. In the present report, while confirming the concept of CD8 T cell programming, we show that this program is flexible and continuously altered by Ag and inflammation, and these two events should be available simultaneously to impart the most beneficial and long-standing program in primed CD8 T cells. Results of the present study do not address whether the program loses flexibility overtime. Current efforts in our laboratory are underway to address this question.

Inflammation is a complex process comprising a series of events leading to innate activation, production of a myriad of cytokines, expression of costimulatory molecules, and up-regulation of MHC. Although our study clearly shows that sustained exposure to inflammation caused by a live infection in association with Ag presentation greatly facilitates clonal expansion and memory differentiation programs of primed CD8 T cells, it does not address the individual components of the inflammation that play a role in this process. Recent studies show that inflammation-mediated third signals play an important role in sustained expansion of CD8 T cells (11, 12, 13). Further studies are needed to address the roles of the specific components of inflammation involved in programming CD8 T cell response in conjunction with Ag.

In summary, our study shows that sustained exposure to Ag and inflammation following initial priming greatly facilitates CD8 T cell expansion and memory differentiation programs, suggesting that the optimal vaccination strategies should attempt to identify and provide the right combination of these two factors for inducing optimal memory responses.


   Acknowledgments
 
We thank C. B. Wilson, M. J. Bevan, P. J. Fink, S. Thomas, L. J. Thompson, and C. Havenar-Daughton for helpful discussions and critical reading of the manuscript, as well as P. Nguyen for technical assistance. We thank P. J. Fink (University of Washington, Seattle, WA) for OT-1 mice.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported in part by National Institutes of Health Grants 1R01AI053146 and R21AI051386 (to K.M.-K.) and by funds from the Washington National Primate Center and the University of Washington Department of Immunology. A.S. is supported by a National Cancer Institute training grant. Back

2 Address correspondence and reprint requests to Dr. Kaja Murali-Krishna, Department of Immunology and Washington National Primate Center, Box 357650 Health Sciences Building, 1959 Northeast Pacific Street, Seattle, WA 98195. E-mail address: mkaja{at}u.washington.edu Back

3 Abbreviations used in this paper: WT, wild type; rLM-OVA, recombinant Listeria monocytogenes expressing OVA; LCMV, lymphocytic choriomeningitis virus; rVSV-OVA, recombinant vesicular stomatitis virus expressing OVA; Lm, Listeria monocytogenes; DC, dendritic cell; Tg, transgenic. Back

Received for publication August 16, 2007. Accepted for publication November 10, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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