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Journal of Virology, February 2007, p. 2002-2011, Vol. 81, No. 4
0022-538X/07/$08.00+0     doi:10.1128/JVI.01459-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Quantitating the Magnitude of the Lymphocytic Choriomeningitis Virus-Specific CD8 T-Cell Response: It Is Even Bigger than We Thought{triangledown}

David Masopust, Kaja Murali-Krishna,{dagger} and Rafi Ahmed*

Emory Vaccine Center and Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, Georgia 30322

Received 10 July 2006/ Accepted 21 November 2006


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ABSTRACT
 
Measuring the magnitudes and specificities of antiviral CD8 T-cell responses is critical for understanding the dynamics and regulation of adaptive immunity. Despite many excellent studies, the accurate measurement of the total CD8 T-cell response directed against a particular infection has been hampered by an incomplete knowledge of all CD8 T-cell epitopes and also by potential contributions of bystander expansion among CD8 T cells of irrelevant specificities. Here, we use several techniques to provide a more complete accounting of the CD8 T-cell response generated upon infection of C57BL/6 mice with lymphocytic choriomeningitis virus (LCMV). Eight days following infection, we found that 85 to 95% of CD8 T cells exhibit an effector phenotype as indicated by granzyme B, 1B11, CD62L, CD11a, and CD127 expression. We demonstrate that CD8 T-cell expansion is due to cells that divide >7 times, whereas heterologous viral infections only elicited <3 divisions among bystander memory CD8 T cells. Furthermore, we found that approximately 80% of CD8 T cells in spleen were specific for ten different LCMV-derived epitopes at the peak of primary infection. These data suggest that following a single LCMV infection, effector CD8 T cells divide ≥15 times and account for at least 80%, and possibly as much as 95%, of the CD8 T-cell pool. Moreover, the response targeted a very broad array of peptide major histocompatibility complexes (MHCs), even though we examined epitopes derived from only two of the four proteins encoded by the LCMV genome and C57BL/6 mice only have two MHC class I alleles. These data illustrate the potential enormity, specificity, and breadth of CD8 T-cell responses to viral infection and demonstrate that bystander activation does not contribute to CD8 T-cell expansion.


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INTRODUCTION
 
The hallmarks of the adaptive immune system are specificity and memory. CD8 T cells recognize peptides presented in the context of major histocompatibility complex (MHC) class I via the T-cell receptor (TCR). Rare naive CD8 T cells that recognize cognate peptide/MHC complexes presented during infection undergo a dramatic activation program. Pathogen-specific CD8 T cells expand, which is coupled to changes in function, phenotype, and trafficking. For instance, activated CD8 T cells upregulate CD44, CD11a, 1B11, and granzyme B, acquire cytolytic activity, downregulate CD62L, and acquire the ability to migrate to nonlymphoid tissues (30). Following pathogen clearance, the activated effector population contracts and differentiates into a phenotypically distinct long-lived resting population known as memory CD8 T cells. Lymphoid memory populations retain effector markers, such as CD44hi and CD11ahi, eventually regain surface CD62L, lose effector molecules such as granzyme B, and undergo a modest rate of cell cycling that does not result in expansion (30, 42). This "homeostatic" proliferation is generally thought to be important for memory CD8 T-cell maintenance. Recent studies suggest that memory T-cell persistence and homeostatic proliferation are antigen-independent (35, 47).

Lymphocytic choriomeningitis virus (LCMV) has been routinely exploited for the study of adaptive immune responses to viral infection. This has led to a relatively thorough characterization of the host response to this natural mouse pathogen. The LCMV genome consists of two ambisense RNA segments that encode a total of four proteins. Examinations of the sequences of the nucleoprotein (NP) and glycoprotein (GP) have identified several potential CD8 T-cell epitopes in C57BL/6 mice, five of which elicit virus-specific cytotoxic T lymphocytes (CTL) during a primary LCMV infection (17, 24, 34, 38, 43, 46, 53).

LCMV infection elicits a 5- to 10-fold increase in the number of CD8+ splenocytes, although original estimates derived from limiting dilution assays suggested that only 1 to 2% of CD8 T cells are specific for LCMV (26, 41). This expansion coincides with an increase in alloreactive and cross-reactive CD8 T cells (36, 56). In addition, infection of LCMV immune mice with murine cytomegalovirus, vaccinia virus, or Pichinde virus causes reactivation of LCMV-specific memory CD8 T cells as detected by bulk CTL assays (45, 57). Injection of mice with microbial products, such as lipopolysaccharide or synthetic double-stranded RNA, induces cell division among a large portion of CD44hi CD8+ T cells (49, 50). This phenomenon depends on type I interferon (IFN-I) and interleukin-15 (IL-15), yet it is antigen independent, as ß2-microglobulin is not required (49, 60). This observation is reminiscent of a report demonstrating that various cytokine combinations could elicit antigen-independent activation and proliferation of naive and memory human CD4 T cells (51). These perplexing phenomena were reconciled by the bystander activation hypothesis; stating that the majority of the CD8 T-cell response following infection is nonspecific expansion driven by the cytokine milieu induced by infection.

This notion precipitated a closer examination of the participants during an ongoing CD8 T-cell response. Several experimental systems demonstrated that naive CD8 T cells neither expand nor express an effector phenotype following heterologous infection or poly(I:C) injection (5, 11, 59). In contrast, memory phenotype CD8 T cells were permissive to bystander activation, as measured by bromodeoxyuridine incorporation, following heterologous infection, treatment with poly(I:C), or exposure to superantigen-activated T cells or {alpha}-galactosylceramide-activated NK T cells (5, 15). These data were consistent with the proposition that bystander activation might play a specialized role in the maintenance of memory CD8 T cells (7). However, other data challenge this view. New tools for detecting antigen-specific CD8 T cells, including MHC I tetramers, intracellular cytokine staining, and enzyme-linked immunospot assay, caused a revision in the frequency estimates of antigen-specific cells (11, 34). Fifty to seventy percent of CD8 T cells at the peak of LCMV infection now appear to be specific for five LCMV-derived epitopes (NP396, GP33, GP276, GP34, and NP205), reducing, but not excluding, potential contributions by bystander activated T cells. Transfer of LCMV-specific memory cells into congenic LCMV carrier mice elicited robust expansion of donor cells but not of host cells, which lack LCMV-specific CD8 T cells (59). In addition, interpretation of the activation of LCMV-specific memory cells by heterologous infections has been complicated by a concern that this phenomenon is based on antigen-dependent cross-reactivity between pathogens (54). Furthermore, a recent report suggests that a portion of LCMV-specific CTL cross-react with unrelated peptides presented by allo-MHC, thus providing an alternative explanation for this line of evidence supporting bystander activation (9).

These observations have led some investigators to conclude that the majority of virus-induced CD8 T-cell expansion is antigen specific (11, 59). However, incomplete identification of all epitopes in any model system has prevented a complete accounting of the specificity of virus-driven expansion, and it remains possible that antigen-independent expansion still contributes significantly to CD8 T-cell responses. Furthermore, recent studies continue to provide data consistent with the notion that bystander expansion contributes to infection-induced CD8 T-cell expansion. For instance, primary human immunodeficiency virus (HIV) infection elicits expansion of CD38+ CD8 T cells. While this is partially due to expansion of HIV-specific precursors, CD38 was also upregulated among CD8 T cells specific for Epstein-Barr virus, cytomegalovirus, and influenza virus (14). A recent report demonstrated that stimulation via CD40, in the absence of exogenous antigen delivery, induced expansion among memory phenotype CD8 T cells (25). Convincing evidence suggests that LCMV infection or other sources of inflammation regulate CD8 T-cell effector function and/or proliferation (15, 21, 27, 28, 37, 55). In light of our failure to fully account for the antigenic specificity of all virus-induced CD8 T-cell expansion, the potential contribution of bystander expansion to antiviral cellular immune responses remains an appealing hypothesis.

As this issue impacts our understanding of the dynamics of antiviral CD8 T-cell responses and may be important to understanding the mechanisms of memory cell maintenance, we explored the contribution of bystander activation to the CD8 T-cell expansion observed upon LCMV infection.


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MATERIALS AND METHODS
 
Mice and infections. Six- to eight-week-old female C57BL/6 (B6) mice were purchased from the National Cancer Institute (Frederick, Maryland). Thy1.1+ P14 mice bearing the H-2Db-GP33-specific TCR were fully backcrossed to B6 mice and maintained in our animal colony (40). Virus stocks of the Armstrong strain of LCMV were plaque purified on Vero cells, grown in BHK-21 cells, and quantitated as previously described (2). Mice were infected by intraperitoneal (i.p.) infection with 2 x 105 PFU of LCMV or by intravenous (i.v.) injection of 1 x 106 PFU of vesicular stomatitis virus (VSV), Indiana serotype (58). P14 chimeric immune mice were generated by adoptively transferring approximately 1 x 105 naive TCR transgenic T cells i.v. into naive B6 mice followed by LCMV infection and 90 days rest. In order to generate secondary memory VSV-specific CD8 T cells, mice originally infected i.v. with 1 x 106 PFU VSV, New Jersey serotype, were rested for three months and infected i.v. with 1 x 106 PFU VSV Indiana (29). All mice were used in accordance with NIH and Emory University Institutional Animal Care and Use Committee guidelines.

Surface staining and tetramers. Single-cell suspensions were prepared, and 1 x 106 cells were stained in phosphate-buffered saline containing 2% bovine serum albumin and 0.1% sodium azide (FACS buffer) for 30 min at 4°C followed by three washes in FACS buffer. Monoclonal antibodies anti-Thy1.1, -Thy1.2, -CD8, -CD44, -CD11a, -1B11, and -CD62L were purchased from BD Biosciences (San Jose, CA), and anti-CD127 was purchased from eBioscience (San Diego, CA). LCMV-specific CD8 T cells were detected using H-2Db tetramers (NP396, GP33, and GP276) and H-2Kd (GP34) tetramers, constructed as previously described (3). VSV-specific CD8 T cells were detected using H-2Kb tetramers containing the N52-59 protein-derived peptide RGYVYQGL (referred to as VSV-N tetramers). Samples were acquired on a FACSCalibur instrument (Becton Dickinson, San Jose, CA). The data were analyzed using FlowJo software (Tree Star, San Carlos, CA).

Tracking cell division with CFSE. Lymphocytes were labeled with CFSE (Molecular Probes, Eugene, OR) as previously described (35). For tracking division of H-2KbN-specific CD8 T cells, 3 x 107 splenocytes from VSV secondary immune B6 (Thy1.2+) mice were labeled with CFSE and transferred i.v. to Thy1.1+ naive B6 mice. For tracking of LCMV-specific memory CD8 T cells, 3 x 107 splenocytes isolated from P14 immune chimeras were labeled with CFSE and transferred i.v. into naive B6 mice.

Intracellular detection of IFN-{gamma}. Splenocytes were cultured for 5 h at a density of 1 x 106 cells/well in a volume of 0.2 ml complete medium in 96-well flat-bottomed plates at 37°C with or without the addition of 0.1 µg peptide. The peptides used were as follows: NP396-404, NP205-212, NP166-175, NP235-243, GP33-41, GP276-286, GP118-125, GP92-101, and GP70-77 (52, 53). Golgiplug (containing brefeldin A; BD PharMingen, San Francisco, CA) was added to stimulated and unstimulated cultures at a dilution of 1 µl/ml. Cells were harvested after 5 h and stained for cell surface antigens. For granzyme B staining, cells were surface stained directly ex vivo, without any in vivo restimulation. After washing in FACS buffer, cells were subjected to intracellular cytokine stain using a Cytofix/Cytoperm kit according to the manufacturer's directions (BD PharMingen). For intracellular staining, we used anti-IFN-{gamma}-APC (BD PharMingen) or anti-human granzyme B-PE (Caltag Laboratories, Burlingame, CA). Cells were fixed in 2% paraformaldehyde/phosphate-buffered saline.


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RESULTS
 
Eighty-five percent of CD8 T cells are granzyme B positive at peak of LCMV response. Infection of immunocompetent C57BL/6 mice with the Armstrong strain of LCMV elicits a transient 10-fold increase in CD8+ splenocytes that peaks eight days following infection (34). This expansion is entirely due to an increase in CD44hi CD8+ T cells (Fig. 1A), which comprise about 95% of CD8+ splenocytes at the peak of the response (Fig. 1B). However, only 50 to 70% of these lymphocytes have been shown to be LCMV specific (data not shown; also see reference 34). As CD44hi is a marker for both activated and memory cells, it is not clear what proportion of these cells are true effector cells (30). To address this question, we examined additional phenotypic markers on CD8+ splenocytes eight days following infection of C57BL/6 mice with LCMV. Granzyme B is a cytolytic molecule typically expressed by effector, but not memory, CD8+ splenocytes (30, 31). As shown in Fig. 1B, only a very small fraction of CD8+ splenocytes isolated from unimmunized mice expressed granzyme B. In contrast, approximately 85% of CD44hi CD8+ splenocytes expressed granzyme B at the peak of the LCMV response.


Figure 1
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  		FIG. 1. Phenotype of CD8+ splenocytes at peak of LCMV response. C57BL/6 mice were infected with 2 x 105 PFU LCMV Armstrong i.p., and splenocytes were analyzed eight days later. (A) Number of CD44lo and CD44hi CD8+ splenocytes isolated from naive and infected mice. (B) Coexpression of granzyme B and CD44 on CD8+ splenocytes isolated from naive or infected mice. (C) Granzyme B expression levels among tetramer-positive versus tetramer-negative CD44hi CD8+ lymphocytes among infected mice. Splenocytes were stained with granzyme B, CD44, CD8, and either NP396, GP34, GP276, or GP33 H-2b tetramers, as indicated. Black line: granzyme B expression among tetramer-negative CD8+ CD44hi splenocytes. Shaded histogram: granzyme B expression among tetramer-positive CD8+ lymphocytes. Thin gray line: isotype control antibody (Ab) staining of tetramer-positive CD8 T cells. (D) Coexpression of granzyme B and various markers of T-cell activation (gated on CD8+ splenocytes). Data in panels A, B, and D are representative of at least three independent experiments (and at least 18 total mice). Data in panel C are representative of two independent experiments (n = 3 per experiment).

Granzyme B staining intensity varied significantly among the CD44hi population (Fig. 1B). Perhaps lower levels of granzyme B expression would distinguish a non-LCMV-specific subset among the expanded CD44hi population. Thus, staining for granzyme B was performed in conjunction with various MHC class I tetramers comprising LCMV immunodominant epitopes: H-2Db/NP396, H-2Db/GP33, H-2Db/GP276, and H-2Kb/GP34. Granzyme B expression among CD44hi tetramer-negative CD8 T cells was equivalent to tetramer-positive cells (Fig. 1C), suggesting that the CD44hi population did not contain an expanded population of granzyme B-negative cells.

CD8+ lymphocytes isolated at the peak of the LCMV response also displayed other phenotypic markers that distinguished them from memory and naive cells (Fig. 1D); 83 to 95% expressed high levels of CD11a and 1B11 and low levels of CD62L and CD127 (4, 18-20, 30). Collectively, these data suggest that the expanded CD44hi CD8+ population present upon LCMV infection is comprised of effector, rather than memory, phenotype cells.

CD8 T cells of unknown specificity mount a robust recall response to LCMV infection and divide >7 times. A cardinal property of memory cells is the ability to mount an antigen-specific recall response upon reinfection. We took advantage of this property in order to assess the participation of bystander activation and expansion in the LCMV response. Naive Thy1.2+ C57BL/6 mice were infected with 2 x 105 LCMV Armstrong i.p. and rested for 3 months. Splenocytes were isolated and 3 x 107 were then transferred to naive Thy1.1+ congenic recipients (Fig. 2A). This system allowed us to distinguish donor cells, containing LCMV-specific memory CD8 T cells, from naive host cells. Recipient mice were then infected with LCMV Armstrong (1 x 106 PFU i.v.), and splenocytes were analyzed seven days later. As seen in Fig. 2B, there was a 40-fold preferential expansion of donor CD44hi CD8+ T cells, relative to host cells. As bystander expansion due to LCMV infection should not discriminate between host and donor cells, these data support the assumption that expansion is primarily antigen specific.


Figure 2
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  		FIG. 2. CD8 T cells of unknown specificity mount a recall response to LCMV infection and divide >7 times. Naive Thy1.2+ C57BL/6 mice were infected with LCMV Armstrong (2 x 105 i.p.). Three months later, splenocytes were isolated and 3 x 107 were transferred into naive Thy1.1+ C57BL/6 mice. Recipients were infected with LCMV Armstrong (1 x 106 PFU i.v.), and spleen was analyzed seven days later. (A) Summary of experimental design. (B) Percentage of CD44hi donor and host CD8+ T cells prior to and seven days following LCMV challenge. (C) Donor (Thy1.2+) and host (Thy1.1+) CD8+ splenocytes were analyzed for coexpression of granzyme B and CD44 or CD11a. (D) Proportion of donor or host CD8 T cells staining with the indicated tetramers. (E) Donor splenocytes were labeled with CFSE prior to transfer in order to track division following infection. Splenocytes were gated on Thy1.2 and CD8, as shown, and CFSE versus CD44 or tetramer staining was analyzed. Data are representative of two independent experiments (n = 3 per experiment).

In accordance with this interpretation, nearly all donor CD8+ lymphocytes expressed granzyme B, compared to 40% of host cells (Fig. 2C). One explanation for the appearance of granzyme B-positive cells among the host population is that priming of LCMV-specific naive CD8 T cells was not completely inhibited by the transfer of memory cells. As shown in Fig. 2D, relatively similar proportions of granzyme B-positive CD8 T cells in both the donor and host populations could be accounted for by staining with four tetramers comprising immunodominant LCMV epitopes (69% of granzyme B-positive donor cells versus 55% of granzyme B-positive host cells). Again, this suggests that both activation (modulation of phenotype) and expansion (increase in cell number) is antigen specific. Nevertheless, we still could not account for all donor cells by tetramer staining with the majority of known LCMV epitopes (Fig. 2D), suggesting that tetramer-negative cells must have expanded as well. To confirm this hypothesis, donor cells were labeled with CFSE before transfer, which allows one to track cell divisions. As seen in Fig. 2E, 98% of donor (CD8+ Thy1.2+) lymphocytes had undergone at least seven divisions. CD44lo donor CD8+ lymphocytes did not divide. As expected, donor lymphocytes specific for LCMV, as determined by tetramer staining, underwent at least seven divisions. Thus far, we have failed to distinguish the proportion of the response known to be specific for LCMV from the component of the response that could not be accounted for in terms of antigen specificity, either by phenotype, degree of expansion, or number of divisions. However, it remains possible that memory CD8 T cells of unique specificity also underwent equivalent division and expansion upon LCMV infection.

Memory cells do not undergo >3 divisions upon heterologous infection. As LCMV infection might induce >7 divisions among memory CD8 T cells in an antigen-nonspecific manner, we examined the effect of LCMV infection on a bona fide memory cell population of unique specificity. Mice were challenged, then rechallenged, with VSV (see Materials and Methods) in order to create a large population of memory CD8 T cells specific for the nucleoprotein of VSV (29). Three months following infection, splenocytes were CFSE labeled and transferred to Thy1.1+ congenic recipients. One day after transfer, recipient mice were infected with LCMV and analyzed eight days later, which corresponds to the peak of the LCMV response (Fig. 3A). If left uninfected, VSV-N-specific donor CD8+ lymphocytes underwent very little homeostatic proliferation over nine days. In contrast, LCMV infection induced one to three divisions among a fraction of VSV-N-specific CD8 T cells. However, this division failed to account for the robust proliferation (>7 divisions) seen among 98% of LCMV immune donor cells upon homologous infection (Fig. 2E).


Figure 3
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  		FIG. 3. Memory CD8 T cells do not divide >3 times upon heterologous infections. (A) Thy1.2+ VSV immune splenocytes (3 x 107) were labeled with CFSE and transferred into Thy1.1+ naive C57BL/6 mice. One day following transfer, recipient mice were either infected with LCMV or left uninfected. (B) Eight days later (peak of LCMV response), the CFSE profile among donor VSV-N tetramer-positive CD8+ T cells was analyzed. (C) Splenocytes (3 x 107) from immune P14 chimeras were labeled with CFSE and transferred to naive B6 mice. One day following transfer, recipient mice were either infected with VSV or left uninfected. (D) Six days later (peak of VSV response), the CFSE profile among donor P14 CD8+ T cells was analyzed (data representative of two independent experiments; n = 3 per experiment). Plots shown are gated on total CD8+ T cells and on Thy1.1+ P14 cells, as indicated.

To confirm this result, we examined the ability of VSV infection to induce proliferation among LCMV-specific memory CD8 T cells. P14 cells are transgenic CD8 T cells specific for GP33 presented in the context of H-2Db (40). Naive Thy1.1+ P14 cells were transferred into naive Thy1.2+ C57BL/6 mice, which were then infected with LCMV. After three months, splenocytes were isolated, labeled with CFSE, and transferred into naive Thy1.2+ mice (Fig. 3C). Recipients were infected with 1 x 106 PFU VSV i.v. and analyzed six days later (the peak of the VSV response). As seen in Fig. 3D, VSV induced one to three divisions among approximately 50% of P14 memory cells. Assuming that no death occurred, this division would result in only a 25.8% ± 1.9% increase in the memory P14 population (i.e., two cells in division "1" represent only a net increase of one cell). In fact, we found no increase in P14 memory CD8 T cells among VSV-infected recipients (7.4 x 103 ± 0.8 x 103) compared to control mice (7.8 x 103 ± 0.2 x 103), suggesting that proliferation may be offset by death. While these data demonstrate that heterologous infection induces bystander proliferation among bona fide memory CD8 T cells of non-cross-reactive antigenic specificities, this division fails to account for perhaps any expansion of CD8+ splenocytes caused by LCMV infection.

Heterologous infection induces transient changes in memory CD8 T-cell phenotype. Our data suggest that bystander activation did not significantly contribute to CD8 T-cell expansion following viral infection. However, it remained possible that heterologous infections induce phenotypic changes on preexisting non-cross-reactive memory CD8 T cells. To examine this issue, we studied the effect of VSV infection on the phenotype of LCMV-specific memory CD8 T cells. P14 chimeric mice were generated as described above, infected with LCMV, and rested for 150 days. Mice were then challenged with VSV. Two days post-VSV infection, we observed a dramatic upregulation of granzyme B and CD69 expression among LCMV-specific memory P14 CD8 T cells (Fig. 4). However, these changes were remarkably transient, and by the peak of the VSV effector CD8 T-cell response, LCMV-specific memory P14 CD8 T cells had reacquired a conventional granzyme B-negative/CD69-negative phenotype. These data indicate that heterologous infections may promote rapid but transient changes in memory CD8 T-cell phenotype.


Figure 4
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  		FIG. 4. Heterologous infection induces transient changes in memory CD8 T-cell phenotype. One hundred fifty days after LCMV infection, P14 chimeric mice were challenged with VSV. Granzyme B and CD69 expression was analyzed on LCMV-specific P14 memory CD8 T splenocytes two and six days after VSV infection and compared to mice that were not infected with VSV. All histograms are gated on Thy1.1+ P14 memory CD8 T cells. The experiment was performed three times (n = 2 to 3 mice per group, per experiment) with similar results.

Eighty percent of CD8 T cells are specific for 10 LCMV epitopes at peak of response. Thus far, our data argue against a contribution of bystander expanded CD8 T cells in the response to LCMV infection. We attempted to provide a more comprehensive analysis of the proportion of the response that was LCMV specific by stimulating effector CD8 T cells with an expanded array of LCMV-derived peptides and performing intracellular staining for IFN-{gamma}. Using this strategy, it was found that nine epitopes were targeted by 80.3% ± 3.9% of CD8 T splenocytes at eight days postinfection (data from 19 mice in five independent experiments; Fig. 5A and B and Table 1). Inclusion of an additional epitope, NP235, accounted for an additional 1.07% ± 0.05% of CD8 T cells (Table 1). It is important to note that the GP33 peptide stimulates both H-2Db/GP33- and H-2Kb/GP34-specific CD8 T cells. Furthermore, rare individual mice had unusually large NP166-specific responses. These same mice had the smallest NP396-specific responses, suggesting a reciprocal relationship in immunodominance between these two epitopes.


Figure 5
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  		FIG. 5. Most CD44hi+ CD8+ lymphocytes are specific for LCMV at peak of infection. Eight (A and B) or 90 (C and D) days following LCMV infection, splenocytes were incubated for five hours with the indicated peptides and brefeldin A, surface stained for CD8 and CD44, permeabilized, and stained with anti-interferon gamma. n = 19 (A and B) or 5 (C and D).


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  		TABLE 1. Summary of LCMV-derived CD8 T-cell epitopes and immunodominancea

Utilization of this expanded epitope repertoire also precipitated a subtle increase in the estimate of LCMV-specific memory cells that persist following infection. It appears that 15.2% ± 0.5% of CD8+ splenocytes are LCMV specific three months following infection (Fig. 5C and D).


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DISCUSSION
 
These experiments demonstrated that at least 80%, and perhaps over 90%, of CD8 T cells are specific for LCMV at the peak of the response (Fig. 2 and 5). LCMV-specific CD8 T cells targeted at least ten different epitopes derived from the NP and GP proteins. Moreover, the expansion in CD44hi CD8 T cells induced by LCMV infection occurs among effector cells that divide >7 times (Fig. 1 and 2). In contrast, VSV- or LCMV-specific memory CD8 T cells only undergo zero to three divisions upon heterologous infection (Fig. 3). Collectively, these data argue strongly against numerically significant contributions of bystander activation to the expansion observed upon LCMV infection. More importantly, these experiments illustrate the potential enormity, breadth, and specificity of virus-specific CD8 T-cell responses.

The frequency of naive GP33-specific CD8 T-cell precursors has been estimated to be 100 to 200/mouse (8). At least 80% of CD8 T cells were specific for LCMV at the peak of the response, which may imply that LCMV-specific CD8 T cells displace naive and/or memory CD8 T cells of alternative specificities. To explore this issue, we compared the total number of CD8 splenocytes to the number of LCMV-specific cells that we identified via IFN-{gamma} staining (Fig. 5). Of course, the numbers of LCMV-specific naive precursors could not be experimentally determined and were estimated by multiplying the number of epitopes analyzed (ten) by the estimated frequency of GP33-specific CD8 T cells. As shown in Fig. 6, there is an approximately 50,000-fold increase (>215) in LCMV-specific CD8 T cells between day zero and day eight postinfection. This expansion was coordinated with an increase in total CD8 splenocytes. Following resolution of infection the LCMV-specific population had contracted significantly, but mice retained a permanent ~1,500-fold increase in the number of LCMV-specific CD8 T cells (combined data from mice 60 to 90 days postinfection). In contrast, the number of "other cells" (those unaccounted for by IFN-{gamma} staining) remained relatively unchanged throughout the response. Thus, the massive increase in LCMV-specific CD8 T cells at the peak of the response can be accounted for by an increase in total CD8 T cells, and it does not appear that LCMV infection induced a very large reduction in CD8 T cells of other specificities.


Figure 6
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  		FIG. 6. LCMV-specific CD8 splenocytes undergo approximately 15 divisions but do not displace CD8 T cells of other specificities during the peak of response. The number of LCMV-specific, non-LCMV-specific, and total CD8 T cells within spleen was quantitated prior to LCMV infection, at the peak of the response, and 60 to 90 days postinfection (memory). The number of naive LCMV-specific CD8 T cells is an estimate based on the reported precursor frequency of GP33-specific CD8 T cells (8).

Our data confirmed the original prediction by Tough et al., that IFN-I-inducing agents, such as viral infection, would elicit bystander proliferation among bona fide LCMV-specific memory CD8 T cells (49), a result previously examined using bromodeoxyuridine incorporation (5). The reciprocal was true in that LCMV infection elicited bystander proliferation among VSV-specific memory CD8 T cells (Fig. 3). This result may appear at odds with the observation that heterologous infection leads to attrition in the memory cell pool (44, 46). However, a report by McNally et al., suggesting that bystander activation was coordinated with an increase in apoptosis, may reconcile these disparate observations (32). In fact, our data demonstrated that bystander division did not result in a net increase among memory CD8 T cells (7.4 x 103 ± 0.8 x 103 P14 in infected recipients compared to 7.8 x 103 ± 0.2 x 103 in control mice), suggesting that bystander proliferation may be balanced with cell death. This conclusion is supported by recent publications by Selin and colleagues (10, 22). They observed that poly(I:C) induced proliferation among bona fide LCMV-specific memory cells but failed to detect expansion of the population. They went on to demonstrate that secondary infections with Pichinde or vaccinia viruses could induce significant division and expansion among a subset of LCMV-specific CD8 T cells, providing compelling evidence that this phenomenon is due to TCR-driven activation via cross-reactive epitopes between pathogens. It will be interesting to determine whether cross-reactivity constitutes a common phenomenon due to homologous peptides between viruses and/or degeneracy of TCR specificity. Nevertheless, these reports are consistent with the notion that bystander activation does not significantly contribute to CD8 T-cell expansion following LCMV infection.

To our knowledge, this study represents the most exhaustive accounting of a polyclonal CD8 T-cell response to date. By employing an expanded array of LCMV-derived peptides and performing intracellular staining for IFN-{gamma}, we were able to directly identify 80% of CD8 T cells as being specific for 10 unique LCMV-derived epitopes (Fig. 5). These data demonstrate not only the incredible magnitude of the CD8 T-cell response to LCMV but also the exquisite specificity of adaptive immune responses. It is also notable that the response to a single virus containing only four proteins is spread among at least 10 different epitopes within mice expressing only two MHC class I alleles. This is all the more striking, considering that only two (the glycoprotein and nucleoprotein) of the four viral proteins have been mined for H-2b binding epitopes (53). It remains likely that the LCMV polymerase and Z protein may contain other epitopes. If so, it might be possible that up to 95% of CD8 T cells in C57BL/6 mice may be LCMV specific eight days after infection.

It will be interesting to learn whether other infections elicit responses to such a diverse array of epitopes. Although this diversity may complicate accurate quantitation of adaptive responses to other infections, it speaks to the incredible robustness of adaptive immunity. Coupled with MHC class I diversity displayed among outbred populations, CD8 T cells may have a large antigenic repertoire to work with, potentially allowing anamnestic responses to infection with phylogenetically related, serologically distinct, and/or novel viruses exhibiting antigenic cross-reactivity. This situation may have positive benefits for protection. For instance, CD8 T cells target common MHC class I restricted epitopes between serologically distinct influenza viruses and may contribute to heterosubtypic immunity (13). Furthermore, it has been proposed that heterologous T-cell immunity could even play a role in protection from novel infections (54). For instance, NP205-, GP33-, and GP92-specific memory CD8 T cells generated to LCMV mount a recall response upon vaccinia virus infection, which correlates with accelerated viral clearance (10, 22, 23). Most importantly, broadly polyclonal T-cell responses would make it less likely that viral mutants could be selected for that avoid cellular immunity due to "holes" in the T-cell repertoire, even in the case of more limited MHC diversity (52). Conversely, numerous specificities of responding CD8 T cells may have negative implications, as well. For instance, dengue virus elicits an acute self-limiting infection that leads to the generation of long-term protective immunity. However, cellular immunity increases one's risk for dengue hemorrhagic fever and dengue shock syndrome upon subsequent infection with unique serotypes (33, 48). It has been noted that a substantial portion of the T-cell repertoire (1 to 10%) may act directly with foreign MHC. As memory cells are more resistant to tolerizing regimens, polyclonal heterologous T-cell immunity may create a barrier to transplantation tolerance. Indeed, it was recently reported that LCMV-specific memory CD8 T cells increase graft rejection in a skin transplantation model (1).

While bystander activation may not lead to CD8 T-cell expansion, heterologous infection may elicit important phenotypic or functional changes among memory cells. For instance, lung infection elicits bystander accumulation of circulating memory cells into the lung airways (16, 39), and coronavirus infection induces bystander trafficking of memory CD8 T cells into the central nervous system (12). Bystander cells also contribute to ocular lesions in a mouse model of herpetic stromal keratitis (6). In addition, primary HIV infection induces a change in the activation status of Epstein-Barr virus-, cytomegalovirus-, and influenza virus-specific CD8 T cells (14). Bystander activation of preexisting memory CD8 T cells may even induce short-term production of IFN-{gamma} and contribute to protection against novel infectious agents independently of TCR stimulation (21, 27, 55). We demonstrated that heterologous infection induced a dramatic upregulation of granzyme B and CD69 expression among memory CD8 T cells, although this was remarkably transient (Fig. 4). Thus, heterologous infection may precipitate changes in homing, phenotype, effector function, and possibly other changes of consequence. Nevertheless, our data suggest that bystander expansion accounts for very little, if any, of the CD8 T-cell expansion induced by LCMV infection.


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ACKNOWLEDGMENTS
 
We thank Vaiva Vezys, Daniel Barber, E. John Wherry, and Barry Rouse for helpful discussions.

This work was supported by a Cancer Research Postdoctoral fellowship (D.M.) and National Institutes of Health grants AI30048 and AI44644 (R.A.).

We have no conflicts of interest.


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FOOTNOTES
 
* Corresponding author. Mailing address: G211 Rollins Research Bldg., Emory University, 1510 Clifton Rd., Atlanta, GA 30322. Phone: (404) 727-3571. Fax: (404) 727-3722. E-mail: ra{at}microbio.emory.edu. Back

{triangledown} Published ahead of print on 6 December 2006. Back

{dagger} Present address: Department of Immunology, University of Washington, Seattle, WA 98195. Back


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Journal of Virology, February 2007, p. 2002-2011, Vol. 81, No. 4
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