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The Journal of Immunology, 2005, 175: 4677-4685.
Copyright © 2005 by The American Association of Immunologists

Recall Proliferation Potential of Memory CD8+ T Cells and Antiviral Protection1

Martin F. Bachmann*, Petra Wolint{dagger}, Katrin Schwarz* and Annette Oxenius2,{dagger}

* Cytos Biotechnology, Zürich-Schlieren, Switzerland; and {dagger} Swiss Federal Institute of Technology, Institute for Microbiology, Zürich, Switzerland


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Memory CD8+ T cells play a crucial role in mediating protection from infection with viruses and other intracellular pathogens. Memory T cells are not a homogenous cellular population and may be separated into central memory T cells with substantial recall proliferation capacity and effector memory T cells with limited recall proliferation capacity. It has been suggested that the protective capacity of effector memory T cells is more limited than that of central memory T cells in viral infections. Here, we show that pronounced recall proliferation potential is indeed key for protection against lymphocytic choriomeningitis virus, which replicates in central lymphoid organs and is controlled by contact-dependent lysis of infected cells. In contrast, recall proliferation competence is not sufficient for protection against vaccinia virus, which is replicating in peripheral solid organs and is controlled by cytokines. To protect against vaccinia virus, high numbers of effector-like T cells were required to be present in peripheral tissue before viral challenge. These data indicate that the protective capacity of different subpopulations of memory T cells may vary dependent on the nature and the route of the challenge infection, which must be considered in T cell-based vaccine design.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The CD8+ T cells play a major role in the control of infections by intracellular pathogens through recognition of pathogen-derived peptides presented on MHC class I molecules. After activation and expansion in secondary lymphoid organs, CD8+ T cells eliminate pathogens by various mechanisms, including lysis of infected cells and secretion of antiviral cytokines or chemokines (1, 2, 3). The relative importance of these effector mechanisms for antiviral control depends on the biology of the individual pathogen. Containment of lymphocytic choriomeningitis virus (LCMV)3 infection is highly dependent on perforin-mediated lysis of infected cells, and secretion of cytokines by CD8+ T cells plays only a minor role (2). In contrast, infection with vaccinia virus is mainly controlled by cytokines such as IFN-{gamma} and TNF-{alpha}, and viral elimination is largely unaffected by the absence of perforin-mediated cell lysis (1, 3). Furthermore, LCMV replication takes mainly place in secondary lymphoid organs, whereas vaccinia virus replication occurs in peripheral organs such as the ovaries and the lung.

Upon virus infection and Ag-specific activation, CD8+ T cells proliferate and differentiate into effector cells. After virus elimination, the CD8+ T cell pool contracts, and the bulk of the specific T cells is eliminated by apoptosis; only ~5–10% of virus-specific cells will enter the memory T cell pool (4, 5, 6, 7, 8, 9). During the effector phase of a T cell response, the survival of precursors of long-lived memory CD8+ T cells is governed by various parameters, including expression of IL-7R{alpha}, CD8{alpha}{alpha}, anti-apoptotic proteins, and protease inhibitors such as Spi2a (10, 11, 12, 13). Furthermore, the presence of virus specific CD4+ T cells during the priming of CD8+ T cell responses is of importance as well, in particular for the generation of memory T cells with pronounced recall proliferation capacities. Specifically, absence of T help (11, 14, 15, 16) but not absence of CD40 (17) induced a population of memory CD8+ T cells that failed to proliferate upon Ag re-exposure but were nevertheless able to home efficiently to infected peripheral tissues after secondary virus challenge.

CD8+ memory T cells are not a homogenous population and can be separated in resting central memory T cells and more activated effector memory T cells (18, 19, 20, 21, 22, 23, 24). Some discrepancies are reported concerning their respective effector functions. Although some reports have demonstrated comparable IFN-{gamma} and TNF-{alpha} production capacities but differences in IL-2 production as well as in their direct cytolytic potential (24, 25, 26, 27), others have found overall differences in cytokine production potential (19, 28, 29).

A hallmark of central memory T cells is their prevalent anatomical location in lymph nodes and their pronounced recall proliferation competence, whereas effector memory T cells fail to undergo significant recall proliferation but efficiently home to peripheral tissues (21, 24, 30, 31, 32). However, in some experimental systems, a pronounced proliferation capacity of effector memory T cells was reported (33, 34, 35). It appears that availability of T help during the priming of CD8+ T cells or during the establishment of memory CD8+ T cell populations is a critical determinant of their recall proliferation potential in some (15, 16, 17, 36) but not all immunization/infection models (37). In line with this, availability of T help was shown to shape the balance between effector and central memory T cell generation (11). An additional factor regulating this balance is the presence of persisting Ag, which is thought to maintain a population of effector memory T cells (38, 39, 40). The issue of recall potential of memory T cell subsets may be further complicated by the observation that the proliferative potential of memory T cells appears to increase over time (35) and differs for effector memory T cells resident in peripheral tissue vs central lymphoid organs (34). Indeed, there may be distinct effector memory T cell populations with distinct ability to proliferate upon antigenic re-exposure.

In the context of T cell-based vaccine development, knowledge about the protective capacity of the different memory CD8+ T cell populations is a crucial requirement. Although still a matter of debate, it is commonly suggested that IL-2-producing, central memory CD8+ T cells exhibiting significant recall proliferation potential represent the major protecting principle (11, 24, 41).

In the present study, we would like to challenge this paradigm. Our results confirm that recall proliferation potential was correlated with protection against systemic LCMV challenge infection. In contrast, protection against vaccinia virus infection was mediated by high numbers of effector memory T cells, which had to be present in the peripheral viral target organs before vaccinia virus challenge.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice, viruses, virus-like particles (VLPs), and peptides

C57BL/6 mice were purchased form Janvier, Ly5.1+ 327 TCR-transgenic mice (42), MHC class II-deficient and CD40-deficient mice were maintained in a specific pathogen-free facility, and mice were immunized between 8 and 12 wk of age. Animal experiments were performed according to the regulations of the cantonal veterinary office.

The LCMV isolate WE was provided by Dr. R. M. Zinkernagel (University Hospital, Zurich, Switzerland) and was propagated at low multiplicity of infection on L929 fibroblast cells. Mice were infected i.v. with 200 PFU of LCMV WE. The LCMV glycoprotein peptide aa 33–41 (gp33 peptide, KAVYNFATM) was purchased from Neosystem.

Recombinant vaccinia virus expressing LCMV glycoprotein (VVG2) was originally obtained from Dr. D. H. L. Bishop (Oxford University, Oxford, U.K.) and grown on BSC40 cells at low multiplicity of infection; quantification was performed as described (43). Mice were infected with 5 x 106 PFU of VVG2 i.p.

gp33-VLPs, based on peptide gp33 coupled to VLPs derived from the bacteriophage Q{beta}, have been described previously (44). Packaging of CpG oligonucleotides (5'-GGGGTCAACGTTGAGGGGGG-3', thioester stabilized) into the gp33-VLPs was performed as described previously (44). Mice were immunized s.c. with 150 µg of gp33-VLPs.

Abs and peptide MHC class I tetramers

Allophycocyanin- or PE-conjugated peptide-MHC class I tetrameric complexes were generated as previously described (45). The following anti-mouse mAbs were purchased from BD Biosciences, BD Pharmingen: anti-CD8 (FITC, PerCP, or allophycocyanin); anti-CD4 (FITC); anti-IFN-{gamma} (PE); anti-TNF-{alpha} (FITC); anti-IL-2 (allophycocyanin).

Immunofluorescent staining and analysis

For direct staining, whole blood or single-cell suspensions from spleens and ovaries were used. Single-cell suspensions from carefully excised ovaries were prepared by gentle dissociation of the tissue through a sterile fine mesh textile fabric using a sterile rubber-coated plunger. Cells were incubated for 20 min at 4°C with peptide-MHC tetramers together with anti-CD8 Abs. Cells were washed and resuspended in PBS containing 1% paraformaldehyde (Sigma-Aldrich). For intracellular cytokine stainings, cells were stimulated with 1 µg/ml gp33 peptide in the presence of brefeldin A for 6 h. Thereafter, cells were surface stained with anti-CD8, fixed and permeabilized as described (27), and stained intracellularly with anti-IFN-{gamma}, anti-TNF-{alpha}, and anti-IL-2 Abs. Four-color flow cytometric analysis was performed using a FACSCalibur flow cytometer (BD Biosciences) with CellQuest software (BD Biosciences). List mode data were analyzed using WinList software (Verity software House).

Viral challenge

gp33-VLP-immunized mice challenged at the indicated time points either i.v. with 200 PFU of LCMV WE or i.p. with 5 x 106 PFU of VVG2. LCMV titers were determined in spleen by the focus-forming assay (46) 3–5 days after challenge. Vaccinia virus titers were determined in the ovaries by plaque assay 1, 2, or 7 days after viral challenge.

51Cr release assay

51Cr release assays were used for the determination of LCMV-gp33-specific cytotoxicity ex vivo on EL4 target cells as described previously (47). In all cases, the starting E:T ratio was adjusted to obtain identical ratios of Db-gp33-specific CD8+ T cells to target cells. Spontaneous release was below 30% in all assays shown.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Short term protection against vaccinia virus challenge in normal and Th-deficient mice

To compare the protective capacity of helped or unhelped LCMV gp33-specific CD8+ T cells at the peak of their expansion, C57BL/6 mice or MHC class II-deficient mice were immunized with bacteriophage Q{beta}-derived VLPs that were modified by covalent coupling of the LCMV-derived peptide epitope gp33–41 (gp33-VLP). To enhance the immunogenicity of the VLPs, they were packaged with CpGs (44). We have previously shown that such CpG-packaged VLPs induce strong Th cell-independent effector CD8+ T cell responses (17, 44). Eight days after priming, gp33-specific T cells had expanded in blood to 6–7% of CD8+ T cells in both C57BL/6 and MHC class II-deficient animals (Fig. 1A). At this time point, animals were challenged i.p. with 5 x 106 PFU of VVG2; 7 days later, gp33-specific T cells had expanded to 20–28% of CD8+ T cells both in blood and in the spleen (Fig. 1, A and B). Upon gp33 stimulation, 30–50% of the gp33-specific CD8+ T cells were able to produce IFN-{gamma}, and the total number of gp33-specific CD8+ T cells did not significantly differ between C57BL/6 and MHC class II-deficient mice. Both helped and unhelped CD8+ T cells were able to confer complete protection against VVG2 challenge and comparable frequencies (and numbers; not shown) of gp33-specific CD8+ T cells were present in the ovaries 7 days after challenge (Fig. 1C).



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  		FIGURE 1. Protection against vaccinia virus by helped and unhelped CD8+ T cells early after immunization. C57BL/6 (B6) and MHC class II-deficient mice were immunized s.c. with 150 µg of gp33-VLPs. Eight days after immunization, mice were challenged i.p. with 5 x 106 PFU of VVG2 (arrow); 7 days later, expansion of gp33-specific CD8+ T cells was analyzed in blood an spleen (A and B) as well as specific IFN-{gamma} production in spleen (B). gp33-specific CD8+ T cell frequencies as well as vaccinia virus titers were measured in the ovaries (C). Each symbol represents the average of at least three mice per group. Error bars indicate the SD within a group.

 
Recall proliferation capacity of CD8+ T cells is gradually lost in Th-deficient mice

In several experimental systems, it was documented that lack of CD4+ T cells during the priming phase of CD8+ T cells led to the development of memory CD8+ T cells which were largely unable to proliferate upon secondary challenge (14, 15, 16, 48), with the notable exception of primary and secondary vesicular stomatitis virus infection, which was Th independent (37). Upon gp33-VLP priming, primary CD8+ T cell expansions were comparable in C57BL/6 and MHC class II-deficient mice, and unhelped gp33-specific CD8+ T cells were still able to proliferate upon secondary in vivo challenge 8 days after priming (Fig. 1). From previous experiments, however, we knew that unhelped CD8+ T cells performed poor recall proliferation by day 40 after gp33-VLP priming (17). We therefore analyzed the kinetics of loss of the in vivo recall proliferation capacity of unhelped CD8+ T cells (Fig. 2). Although helped CD8+ T cells consistently showed significant secondary expansion after in vivo VVG2 challenge, secondary expansion of unhelped CD8+ T cells gradually declined over time. Significant secondary proliferation was apparent until ~20 days after gp33-VLP priming. After day 30, however, secondary proliferation of unhelped CD8+ T cells was lost.



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  		FIGURE 2. A, Unhelped CD8+ T cells gradually lose their proliferation competence after secondary challenge with VVG2. C57BL/6 (B6) and MHC class II-deficient mice were immunized s.c. with 150 µg of gp33-VLPs. From 8 to 55 days (d) after immunization, mice were challenged i.p. with 5 x 106 PFU of VVG2, and gp33-specific CD8+ T cell frequencies were determined in blood before (•) and 7 days after ({circ}) VVG2 challenge. Each symbol represents the average of a least three mice per group. In the bottom panel, the relative expansion of gp33-specific CD8+ T cells is shown for B6 and MHC class II-deficient mice up to day 34 after gp33-VLP immunization. B, 105 TCR-transgenic CD8+ T cells were adoptively transferred into C57BL/6 or MHC class II-deficient hosts and immunized with 150 µg of gp33-VLPs. From 8 to 48 days after immunization, CD62L and CD127 expression was analyzed on gp33-specific CD8+ T cells. Each symbol represents the average of at least three mice per group. tet, Tetramer.

 
Next, we longitudinally phenotyped gp33-specific CD8+ T cells after gp33-VLP immunization in B6 or MHC class II-deficient mice by L-selectin (CD62L) and CD127 expression. The low frequencies of endogenous gp33-specific CD8+ T cells after 3 wk of immunization precluded a clear phenotypical analysis. We therefore chose to adoptively transfer low numbers of gp33-specific TCR-transgenic CD8+ T cells into C57BL/6 or MHC class II-deficient mice before immunization with gp33-VLPs. In MHC class II-deficient hosts, a majority of gp33-specific CD8+ T cells maintained a CD62L–CD127– phenotype up to 48 days after immunization; whereas in C57BL/6 hosts, a majority of cells were CD62L+CD127+ at 48 days after immunization (Fig. 2B). Thus, unhelped memory CD8+ T cells exhibited characteristics of effector memory T cells in terms of both recall potential and expression of cell surface markers.

Recall proliferation capacity of memory CD8+ T cells fails to confer long term protection against vaccinia virus infection

To assess whether recall proliferation capacity of CD8+ T cells was a hallmark for antiviral protection, we compared the protective capacity of helped and unhelped gp33-VLP-primed CD8+ T cells longitudinally (Fig. 3). C57BL/6 and MHC class II-deficient mice were immunized with gp33-VLPs; 8–55 days later, mice were challenged with VVG2. Already at day 19 after immunization, both helped and unhelped CD8+ T cells controlled viral infection only partially; by day 55 after immunization, no protection could be observed, despite significant expansions of helped gp33-specific CD8+ T cells in blood (Fig. 3) and spleen (not shown) and despite the presence of large frequencies of helped and unhelped gp33-specific CD8+ T cells in the ovaries 7 days after challenge (Fig. 3B). Although we have previously seen that VLP priming does not confer long term protection (49), we were still surprised how inefficient protection mediated by the helped T cells was, because viral titers were measured 7 days after challenge infection. At this late time point, even naive mice start to control viral infection. Thus, recall proliferation capacity of CD8+ T cells is clearly no correlate of protection in vaccinia virus challenge infection.



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  		FIGURE 3. Helped and unhelped CD8+ T cells gradually lose their protective capacity against secondary challenge with VVG2. C57BL/6 (B6; •) and MHC class II-deficient mice ({circ}) were immunized s.c. with 150 µg of gp33-VLPs. From 8 to 55 days after immunization, mice were challenged i.p. with 5 x 106 PFU of VVG2, and gp33-specific CD8+ T cell frequencies were determined in spleen and ovaries either before or 7 days after VVG2 challenge (B). Representative FACS stainings of gp33-tetramer (tet)+ CD8+ T cells in ovaries are shown for mice challenged 22 days (d) after VLP priming (A). Vaccinia virus titers were determined in ovaries at the same time point (C). Each symbol represents the average of at least three mice per group.

 
Peripheral presence of CD8+ T cells is required for protection against vaccinia virus

From these experiments we concluded that strong recall responses of gp33-specific CD8+ T cells upon VVG2 challenge and hence the presence of high numbers of gp33-specific CD8+ T cells in the ovaries 7 days after VVG2 challenge were rather a result of local vaccinia virus replication than a correlate of protection. We therefore hypothesized that the presence of gp33-specific CD8+ T cells before VVG2 challenge might correlate with antiviral protection. To test this hypothesis, C57BL/6 mice were immunized with gp33-VLPs, and 8 or 27 days later mice were challenged with 5 x 106 PFU VVG2. Frequencies of gp33-specific CD8+ T cells were measured in the ovaries at the time of challenge or 1, 2, 3, and 5 days after challenge. Viral titers were determined 3 and 5 days after challenge (Fig. 4). Indeed, antiviral protection clearly correlated with the early presence of large numbers of gp33-specific CD8+ T cells in the ovaries; in fact, it correlated with the presence of significant numbers of gp33-specific CD8+ T cells before VVG2 challenge. Eight days after gp33-VLP priming, ~35% of the CD8+ T cells in the ovaries were gp33 specific, corresponding to roughly 1300 gp33-specific CD8+ T cells per ovary. These frequencies were largely maintained during the first 5 days after VVG2 challenge, and the vast majority of these cells were able to produce IFN-{gamma} and TNF-{alpha} upon gp33 stimulation (Fig. 4A). Even before VVG2 challenge, frequencies of gp33-specific CD8+ T cells were much higher in the ovaries compared with blood and spleen, indicating that effector (memory) CD8+ T cells are preferentially migrating to and residing in peripheral tissue. Moreover, frequencies of gp33-specific CD8+ T cells showed a transient decline in spleen 1 day after VVG2 challenge, suggesting that some redistribution to sites of Ag replication might have occurred. In comparison with frequencies on day 8 after gp33-VLP priming, frequencies of gp33-specific CD8+ T cells had significantly declined in blood, spleen, and also in the ovaries by day 27 after priming (Fig. 4B). Within the first 2 days after VVG2 challenge, no significant increase in gp33-specific CD8+ T cell frequencies was seen in the infected ovaries, explaining the minimal protection against VVG2 challenge in these mice. However, by day 3 after challenge, gp33-specific CD8+ T cells had massively expanded in spleen and had infiltrated the infected ovaries in large frequencies.



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  		FIGURE 4. VVG2 protection is conferred by gp33-specific CD8+ T cells in ovaries before VVG2 challenge. C57BL/6 mice were immunized s.c. with 150 µg of gp33-VLPs. Eight (A) or 27 (B) days after immunization, mice were challenged i.p. with 5 x 106 PFU of VVG2 (arrow) and gp33-specific CD8+ T cell frequencies were determined in the ovaries and the spleen either before or 1, 2, 3, or 5 days after VVG2 challenge. The frequency of CD8+ T cells producing IFN-{gamma} and TNF-{alpha} after in vitro gp33 stimulation was also assessed in ovaries and spleen 1 or 2 days after VVG2 challenge. Vaccinia virus titers were determined in the ovaries 3 and 5 days after challenge of VLP-primed mice (•) and of naive control mice ({circ}). Each symbol represents the average of at least three mice per group.

 
Thus, the presence of significant numbers of gp33-specific CD8+ T cells in ovaries before infection causes an early reduction of viral titers and thus leads to protection. In contrast, at later time points after immunization, the absence of gp33-specific CD8+ T cells in the peripheral viral target organs in combination with delayed infiltration into these sites resulted in impaired protection from viral replication.

Short term protection against LCMV challenge in normal and Th-deficient mice

In analogy to the previous experiments with VVG2 challenge, we next tested the protective capacity of helped and unhelped gp33-VLP induced CD8+ T cells against LCMV challenge. C57BL/6 or MHC class II-deficient mice were immunized with gp33-VLPs and challenged at the peak of the primary response with 200 PFU LCMV. Five days after LCMV challenge, frequencies of gp33-specific CD8+ T cells were measured in blood and spleen (Fig. 5, A and B). Frequencies of gp33-specific CD8+ T cells had declined in blood, most likely due to redistribution to sites of viral replication. Helped and unhelped gp33-specific CD8+ T cells in the spleen were equally functional with respect to IFN-{gamma}, TNF-{alpha}, and IL-2 secretion (Fig. 5B) and with respect to cytolytic activity (Fig. 5C). Moreover, comparable total numbers of helped and unhelped gp33-specific CD8+ T cells were present in spleen (Fig. 5B), and all groups of gp33VLP-primed mice had completely eliminated LCMV from the spleen by day 5 after challenge infection.



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  		FIGURE 5. Protection against LCMV by helped and unhelped CD8+ T cells early after immunization. C57BL/6 (B6) mice and MHC class II-deficient mice were immunized s.c. with 150 µg of gp33-VLPs. Eight days after immunization, mice were challenged i.v. with 200 PFU of LCMV (arrow); 5 days later, expansion of gp33-specific CD8+ T cells was analyzed in blood an spleen (A and B) together with specific IFN-{gamma}, TNF-{alpha} and IL-2 production in spleen (B). % of CD8 refers to the percentage of gp33-tetramer (tet)+ or cytokine-producing cells of CD8+ T cells; total number/spleen refers to the total number of gp33-tet+ or cytokine-producing CD8+ T cells per spleen. C, Direct ex vivo cytotoxicity was measured in a 14-h 51Cr release assay. E:T ratio is the ratio between gp33-specific CD8+ T cells and gp33-loaded (•) or mock-loaded ({circ}) EL4 target cells. Infectious LCMV titers were determined in spleen by focus-forming assays (C). Each symbol represents the average of at least three mice per group.

 
Recall proliferation capacity of memory CD8+ T cells is associated with protection from LCMV infection

To test the importance of recall proliferation capacity of memory CD8+ T cells for their long term protective potential against LCMV, C57BL/6, or MHC class II-deficient mice were immunized with gp33-VLP and challenged with LCMV between 8 and 55 days after priming (Fig. 6). As observed for vaccinia virus challenge, both unhelped and helped CD8+ T cells were able undergo secondary proliferation after LCMV challenge up to 3 wk after VLP priming. At later time points, however, CD8+ T cells strongly expanded in C57BL/6 mice upon booster infection with LCMV, whereas no such expansion was seen in MHC class II–/– mice (Fig. 6). The protective potential of the T cells was assessed by measuring LCMV titers 5 days after infection. Eight and 22 days after priming, both helped and unhelped CD8+ T cells completely eliminated LCMV within 5 days after infection. However, starting at day 24 after priming, unhelped CD8+ T cells gradually lost their protective potential (Fig. 6B) concomitant with their loss of recall proliferation capacity (Fig. 6A). These findings show that, in contrast to VVG2 challenge, long term protection against LCMV challenge is linked to memory CD8+ T cells with significant recall proliferation capacity and is therefore dependent on helped CD8+ T cells.



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  		FIGURE 6. Unhelped CD8+ T cells gradually lose their protective potential against secondary challenge with LCMV. C57BL/6 and MHC class II-deficient mice were immunized s.c. with 150 µg of gp33-VLPs. From 8 to 55 days after immunization, mice were challenged i.v. with 200 PFU of LCMV, and gp33-specific CD8+ T cell frequencies were determined in blood and spleen 5 days (d) after LCMV challenge (A). LCMV titers were determined in spleen at the same time point (B). Each symbol represents the average of at least three mice per group.

 
LCMV protection by memory CD8+ T cells is delayed

Interestingly, control of LCMV infection by memory CD8+ T cells was not immediate, because infectious LCMV could still be detected early after LCMV challenge infection (day 3) but not at a later time point (day 5) in C57BL/6 mice which had been primed with gp33-VLPs 24 days previously. When gp33VLP-primed C57BL/6 mice were challenged at day 24 with an intermediate dose of LCMV (104 PFU), high viral titers were detected in the spleen 3 days after challenge, but already by day 5 after challenge, these had dropped by >2 logs (Fig. 7D). Even a low dose (200 PFU) of LCMV replicated to a sizable titer in the spleen within 3 days but was efficiently controlled by day 5 (Fig. 7D). The failure of unhelped memory CD8+ T cells to control LCMV infection was not due to a lack of effector function, because ex vivo lytic activity on a gp33-specific cell basis was comparable for helped and unhelped memory CD8+ T cells (Fig. 7C). However, the failure of unhelped CD8+ T cells to confer protection against LCMV challenge was due to insufficient expansion between day 3 and 5 after viral challenge in blood and in spleen (Fig. 7, A and B).



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  		FIGURE 7. LCMV protection is not immediate. C57BL/6, MHC class II-deficient and CD40-deficient mice were immunized s.c. with 150 µg of gp33-VLPs. Twenty-four days after immunization, mice were challenged i.v. with 200 PFU (or 104 PFU; D) of LCMV (arrow), and gp33-specific CD8+ T cell frequencies were determined 3 and 5 days later in blood (A) and spleen (B). Frequencies of CD8+ T cells, producing IFN-{gamma}, TNF-{alpha}, and IL-2 after in vitro gp33 stimulation, were determined in spleen (B). Direct ex vivo cytotoxicity was measured 5 days after LCMV challenge by 6-h 51Cr release assays (C). E:T ratio is the ratio between gp33-specific CD8+ T cells and gp33-loaded (•) or mock-loaded ({circ}) EL4 target cells. Infectious LCMV titers were determined in spleen by focus-forming assays 3 and 5 days after challenge with either 200 PFU or 104 PFU of LCMV (D). Each symbol represents the average of at least three mice per group.

 
CD40L has been proposed to be a key molecule for the development of memory CD8+ T cells with recall proliferation capacity (11, 50). We addressed the importance of CD40-CD40L interaction by testing the consequences of lacking CD40-CD40L interactions in our VLP-priming/LCMV challenge model (Fig. 7). CD40-deficient mice were immunized with gp33-VLPs and challenged 24 days later with LCMV. For all analyzed parameters (recall expansion, cytokine production, cytolytic activity, and antiviral protection), CD40-deficient mice were comparable with normal C57BL/6 mice, demonstrating that CD40-CD40L interactions were dispensable for the induction of memory CD8+ T cells with recall proliferation capacity (17) as well as for their protective potential against LCMV challenge.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The relative importance of central memory T cells (mostly exhibiting significant recall proliferation capacity) vs effector memory T cells (mostly exhibiting limited recall proliferation capacity) in mediating protective immunity is a matter of debate. Although some studies concluded that activated or effector memory T cells were more potent at mediating protection against peripheral viral infections (18, 20, 33, 34, 51, 52, 53), other reports suggested that central rather than effector memory T cells mediate protection (11, 24, 41). Here, we demonstrate that the requirement of recall proliferation capacity for antiviral protection by memory T cells, induced by vaccination with replication-incompetent VLPs, varies with the viral challenge model studied. Although low numbers of memory CD8+ T cells exhibiting recall proliferation capacity were sufficient for protection against LCMV replication in the spleen, high numbers of effector memory T cells residing in peripheral organs at the time of infection were required for protection against replication of vaccinia virus in ovaries.

A possible explanation for the difference may be the distinct biology of LCMV and vaccinia virus. An important difference between the viruses is the site of replication; LCMV replicates primarily in lymphoid organs, whereas vaccinia virus replicates in nonlymphoid peripheral organs, such as the ovaries. A second important difference is that cytokines, in particular IFN-{gamma} and TNF-{alpha}, mediate protection against vaccinia virus (1, 54), whereas lysis of infected cells is the primary mechanism of protection against infection with LCMV. Thus, for control of LCMV, high numbers of specific CD8+ T cells are required, because these cells must engage individual infected cells in the spleen and eliminate these target cells before large quantities of virus progeny has been released. In the case of LCMV, viral replication appears to occur sufficiently slowly to allow central memory T cells to differentiate and accumulate to sufficiently high numbers for control of LCMV infection. Even if few memory cells were present and high doses of virus were used for challenge infection, memory T cells expanded sufficiently rapid to cause complete viral elimination at a time point when naive mice only just started clearing the virus (55). A different situation applies to vaccinia virus challenge. Here, protection from infection occurs through cytokines and direct contact between infected cells and specific T cells is not required. Yet, memory T cells lose the race against rapid viral replication unless they are present at the site of peripheral infection before infection occurs. Thus, restimulation and secondary expansion of central CD8+ memory cells in secondary lymphoid organs followed by migration of activated cells to peripheral sites of infection is too slow for efficient control of a peripheral viral infection. These findings are reminiscent of those obtained with secondary influenza or Sendai virus infection, in which the presence of significant numbers of T cells at sites of peripheral viral challenge correlated with protection (33, 34, 52, 53). Thus, these results suggest that long term protection against a viral challenge infection taking place in lymphoid organs and being controlled by contact-dependent cell lysis may require central memory T cells whereas long term protection against a peripheral viral challenge that is mediated by T cell-secreted cytokines requires significant number of effector memory T cells present at the site of viral challenge.

These findings have potential implications for vaccine design. Induction of conventional long-lived central memory T cells by vaccination may not suffice to protect against infection with many viruses, including influenza and pox viruses. However, such vaccines may still facilitate accelerated elimination of viruses such as LCMV, which mainly replicate in lymphoid organs and are cleared by lysis of infected cells. HIV may be such a virus because it replicates predominantly in lymphoid organs during chronic infection. Indeed, vaccination of macaques, using DNA or recombinant vaccinia virus, led to reduced peak viremias and reduced viral set points after systemic SIV challenge (56, 57). However, natural HIV entry occurs mainly at peripheral sites and absence of HIV-specific T cells at these peripheral entry sites, such as the intestinal tissue, might leave enough time for the virus to establish a prominent infection which is subsequently more difficult to control. Thus, although the development of a vaccine against HIV has yielded rather disappointing results thus far, current efforts in developing strong immunogens with the potential of inducing large numbers of central and effector T cells may still result, at least, in a modulation of the course of the infection.


   Acknowledgments
 
We thank Urs Karrer for critical reading of the manuscript.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
M. F. Bachmann and K. Schwarz are Cytos employees and own stocks or stock options of the company.


   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 by the Roche Research Fund for Biology, the Swiss National Science Foundation, and the Vontobel Foundation. Back

2 Address correspondence and reprint requests to Dr. Annette Oxenius, Swiss Federal Institute of Technology, Institute for Microbiology, Eidgenössiche Technische Hochschule Hönggerberg, Wolfgang-Pauli-Strasse 10, HCI G401, 8093 Zürich, Switzerland. E-mail address: oxenius{at}micro.biol.ethz.ch Back

3 Abbreviations used in this paper: LCMV, lymphocytic choriomeningitis virus; VLP, virus-like particle; VVG2, recombinant vaccinia virus expressing LCMV glycoprotein; CD62L, L-selectin. Back

Received for publication May 25, 2005. Accepted for publication July 26, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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