Following intranasal administration, the model paramyxovirus simian virus 5 (SV5) establishes an infection in the respiratory tract of mice, which is subsequently cleared by CD8+ T cells. In this study, we sought to understand the maturation of the antiviral immune response over time by assessing the functional avidity of the responding T cells and the expansion of immunodominant populations. Surprisingly, we determined that the initial response to Ag at day 3 (d3) in the mediastinal lymph node was exclusively high avidity. However, by d5 postinfection, low avidity cells were ∼50% of the responding T cell population. Following secondary exposure to SV5, high avidity CD8+ T cells again are the exclusive cell type present at early times postinfection (d2). Similarly, high avidity cells were preferentially elicited at d3 following infection with the unrelated vaccinia virus. We also made the observation that the immunodominance profile has not been established at d3 postinfection with SV5. However, by d5 a clear immunodominance pattern arises and is permanently maintained. These data indicate that high avidity cells are the predominant population responding at early times postinfection following respiratory infection with SV5 or vaccinia virus. However, as the response progresses, low avidity cells are activated/expanded to a greater extent compared with high avidity cells.

CD8+ T cells have been shown to contribute significantly to the optimal clearance of many respiratory pathogens, i.e., Sendai virus and influenza virus (1, 2). However, questions remain concerning the attributes of the antiviral CD8+ T cell responses that confer optimal protection.

We have used simian virus 5 (SV5)3 as a model system to study the immune response following viral infection in the respiratory tract (3). SV5 has long been considered a prototypic member of the paramyxoviridae family of viruses whose members include common respiratory pathogens such as respiratory syncytial virus, mumps virus, and the human parainfluenza viruses 1–4. Members of this family of viruses have nonsegmented negative strand RNA genomes. The ∼15-kb SV5 RNA genome consists of seven tandemly linked genes that encode eight different proteins. The small number of viral proteins expressed by SV5 allows us to easily follow the immune response elicited by infection with the virus.

Of the many peptides encoded by a complex Ag, such as a viral particle, that can potentially be presented and elicit a CD8+ T cell response, only a limited number do so (for review, see Ref. 4). Within this small fraction often a single epitope elicits the immunodominant response, while the remaining immunogenic epitopes elicit subdominant immune responses (4, 5, 6). Although the mechanisms that contribute to the emergence of immunodominance are not fully understood, a number of factors have been shown to contribute, at least in part, to the establishment of immunodominance, including peptide affinity and the level of presentation at the cell surface (7). It has also been shown that immunodominance is flexible and is relative for a given set of immunogenic epitopes. For example, following loss of an immunodominant epitope, the response to subdominant epitopes is able to compensate for the loss of the immunodominant determinant (8, 9). There is significant interest in elucidating the mechanisms involved in controlling immunodominance, as it has been suggested to have a role in immune escape and delayed clearance as a result of the intense focusing of the immune response toward a single epitope.

Although CD8+ T cells have a well-documented role in the clearance of viral infections, not all Ag-specific T cells are equivalent in their ability to reduce viral load or protect from challenge. CD8+ T cell avidity is one characteristic that influences the efficacy of a given cell. We and others have demonstrated the in vivo importance of high avidity CD8+ T cells (10, 11, 12). In adoptive transfer studies, high avidity cells were much more effective than low avidity cells in reducing viral load. The reports to date have used two models: vaccinia virus delivered i.p. (10) and lymphocytic choriomeningitis virus administered intracerebrally (12). To our knowledge, no studies have evaluated the avidity of responses to viral infections in the respiratory tract, the site of entry for the majority of viruses.

Little is known about the in vivo regulation of high vs low avidity T cells. For example, the parameters promoting the activation and expansion of high and low avidity cells and the kinetics with which they respond to viral infection are not known. In addition, many questions remain in our understanding of the mechanisms by which immunodominance is established and maintained. In an effort to understand the regulation of the antiviral CD8+ T cell response from its initiation to the establishment of memory, immunodominance hierarchies and functional avidity were analyzed in BALB/c mice during the course of a respiratory tract infection with SV5. The findings from these studies have important implications for our understanding of the antiviral response elicited following respiratory tract infection.

BALB/c mice were purchased from the Frederick Cancer Research and Development Center (Frederick, MD). P815 is a DBA/2-derived (H-2d) mastocytoma. All research performed on mice in this study complied with federal and institutional guidelines set forth by the Wake Forest University Animal Care and Use Committee. Recombinant viruses were constructed, as previously described (3), using the W3 SV5 strain or the WR vaccinia virus strain.

Mice were anesthetized with Avertin (2,2,2-tribromoethanol) by i.p. injection. Anesthetized mice were then immunized intranasally (i.n.) with the indicated amount of virus in 50 μl of PBS.

Lymphocytes from the lung were isolated from infected mice, as previously described (13). Briefly, lungs were extracted and pooled from two to four mice, finely chopped, and incubated at 37°C for 20 min in 10 ml of digestion mixture. The digestion mixture consisted of RPMI 1640, 10% FCS, 1 mg/ml of collagenase D (Roche), and 20 μg/ml DNase I type IV from bovine pancreas (Sigma-Aldrich, St. Louis, MO). Following digestion, the tissue was pushed through a nylon cell strainer and centrifuged over a Ficoll gradient (Sigma-Aldrich) to isolate mononuclear cells. Mediastinal lymph nodes (MLN) and spleens were isolated and pooled from two to four mice that had been immunized as indicated. Single cell suspensions were made.

The ELISPOT assays were performed, as previously described (3). Briefly, responder cells isolated from the spleen, MLN, or lungs of mice immunized with either wild-type (WT) rSV5 or recombinant vaccinia viruses (VV) expressing SV5 polypeptides were cocultured with P815 stimulators that had been infected with the indicated virus 18–24 h prior and then UV inactivated. Because P815 cells express MHC class I Ags and not MHC class II Ags, the IFN-γ production observed was mediated through class I-restricted T cells. To enumerate the numbers of high and low avidity cells, titrated numbers of responders were cultured in the presence or absence of saturating concentrations (as determined by flow cytometric analyses) of anti-CD8 Ab (clone 53 6.72) in the form of ascites. Following 36–48 h of coculturing of responder and stimulator cells at 37°C, the plates were developed, as described previously (3). The number of spots was determined with the aid of a stereoscope. The relative avidity of cells was determined by the differential sensitivity of high vs low avidity CD8+ T cells to blocking with anti-CD8 Ab. Higher avidity T cells produce IFN-γ in the presence of anti-CD8-blocking Ab following stimulation by APC infected with virus, and T cells of lower avidity are blocked by anti-CD8 Ab (3, 14, 15). Nonspecific spot production was assessed by culturing the highest input number of responder cells in the presence of stimulator cells that were uninfected or were infected with viruses expressing irrelevant Ags (i.e., the HIV glycoprotein, gp160). The number of spots in the absence of infection was negligible.

Peptide ELISPOT assays were performed by incubating P815 stimulators with titrated doses of M285–293 (IPKSAKLFF) peptide at 37°C for 3 h. Stimulator cells were then washed and cultured with T cells from the MLN of immunized mice in an ELISPOT assay. As a negative control, T cells were cocultured with P815 cells that had not been loaded with peptide.

In an effort to further understand the response to respiratory infection with SV5, kinetic experiments were performed to study the emergence of the SV5-specific response following i.n. immunization. Responder cells from the MLN, spleen, and lung were isolated at different time points postinfection and analyzed by ELISPOT assay. Stimulators for this assay were P815 cells infected with vaccinia viruses expressing individual SV5 proteins. Importantly, P815 cells do not express MHC class II, and therefore only class I-restricted CD8+ T cells are stimulated to produce IFN-γ. At day 3 (d3) postinfection, SV5-specific CD8+ effector T cells were detected in the MLN, but not the spleen or lung (Fig. 1). The presence of cells in the MLN, but not the spleen, is expected given i.n. delivery of the virus. No SV5-specific cells were detected at d2 postinfection (data not shown). Significant expansion of T cells occurred in the MLN between d3 and d7 with a subsequent decline by d12. The peak of the SV5-specific response at d7 is similar to that reported for i.n. infection with influenza virus (16).

FIGURE 1.

The CTL response to i.n. infection with SV5 in BALB/c mice is initiated in the MLN. BALB/c mice were immunized i.n. with 1 × 106 PFU of WT rSV5, and CD8+ T cells from the spleen (•), MLN (▪), and lung (▴) were isolated for analysis by ELISPOT. Total SV5-specific spots at each time point were calculated by subtracting the background out of each SV5 protein-specific response and adding them together. Each time point represents the average of three or four separate experiments.

FIGURE 1.

The CTL response to i.n. infection with SV5 in BALB/c mice is initiated in the MLN. BALB/c mice were immunized i.n. with 1 × 106 PFU of WT rSV5, and CD8+ T cells from the spleen (•), MLN (▪), and lung (▴) were isolated for analysis by ELISPOT. Total SV5-specific spots at each time point were calculated by subtracting the background out of each SV5 protein-specific response and adding them together. Each time point represents the average of three or four separate experiments.

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The responses in the spleen and the lung were delayed compared with the MLN. By d5, SV5-specific CD8+ cells were detected in the spleen; however, the absolute numbers of Ag-specific CD8+ T cells were significantly lower than that detected in the MLN. Once the splenic response peaked during the acute phase of the response at d7, the relative size of the response remained similar over time. Only very low numbers of CD8+ T cells could be detected in the lung before d7. The appearance of larger numbers of CD8+ T cells in the lung correlates with the clearance of viral Ag from the lung, as measured by Western blot of lung tissue (data not shown). The number of CD8+ T cells continued to increase in the lung through d12.

Analysis of the memory response (d40) demonstrated that the majority of memory cells were found to reside in the spleen. This is in agreement with the results reported in other systems (17). Furthermore, SV5-specific cells were detectable in the lung, but as would be expected, the number was much smaller than at d12.

We have previously shown that the immunodominant response to SV5 in BALB/c mice is directed against an epitope in the M protein, whereas subdominant responses are directed against epitopes in the P, F, and HN proteins (3). To address the emergence of T cells of high vs low avidity over time, we performed a kinetic analysis of the antiviral CD8+ T cell response in BALB/c mice immunized i.n. with SV5. On the indicated days postinfection, responder cells from the MLN were isolated and tested in an ELISPOT assay for CD8+ T cells that recognize the SV5 P, M, F, or HN proteins. Our assay for T cell avidity is based on the observation that low avidity cells require CD8 engagement to elicit effector function, whereas high avidity cells are relatively CD8 independent for activation (3, 15, 18, 19, 20, 21, 22). As previously described, we were able to quantify the number of high and low avidity CTL specific for SV5 epitopes by including anti-CD8-blocking Ab in cocultures of responding T cells and stimulators (P815 cells infected with VV-P, VV-M, VV-F, or VV-HN) (3). Only those CTL that are of high avidity will produce IFN-γ in the presence of anti-CD8-blocking Ab (filled bars). Cultures incubated in the absence of Ab detect the total response to each protein (hatched bars). The number of low avidity cells (CD8 dependent) can be determined by subtracting the number of cells detected in the presence of anti-CD8 Ab from the total response.

Surprisingly, in the MLN at d3 postinfection, the response to each of the SV5 proteins was almost exclusively high avidity (Fig. 2 A; compare hatched bars with filled bars). However, by d5 postinfection, low avidity cells were readily detected and comprised ∼50% of the total responding CD8+ T cells. In agreement with our previous work (3), the P-specific response remained exclusively high avidity.

FIGURE 2.

High avidity T cells are selectively activated or expanded at early times postinfection with SV5. BALB/c mice were immunized i.n. with 1 × 106 PFU of WT rSV5, and MLN were isolated and pooled from four mice for each time point. ELISPOT assays were performed (A) in the absence (hatched bars) or presence (filled bars) of anti-CD8-blocking Ab. P815 cells were infected with recombinant vaccinia viruses expressing the SV5 P, M, F, and HN proteins, and were cocultured with MLN populations to stimulate production of IFN-γ. As a negative control, the HIV glycoprotein, gp160, was used. B, BALB/c mice were immunized, and at d3 (filled symbols) and d7 (open symbols) postinfection cells from the MLN were isolated and analyzed by ELISPOT assay with titrated doses of M285–293 peptide. The data shown are representative of three independent experiments.

FIGURE 2.

High avidity T cells are selectively activated or expanded at early times postinfection with SV5. BALB/c mice were immunized i.n. with 1 × 106 PFU of WT rSV5, and MLN were isolated and pooled from four mice for each time point. ELISPOT assays were performed (A) in the absence (hatched bars) or presence (filled bars) of anti-CD8-blocking Ab. P815 cells were infected with recombinant vaccinia viruses expressing the SV5 P, M, F, and HN proteins, and were cocultured with MLN populations to stimulate production of IFN-γ. As a negative control, the HIV glycoprotein, gp160, was used. B, BALB/c mice were immunized, and at d3 (filled symbols) and d7 (open symbols) postinfection cells from the MLN were isolated and analyzed by ELISPOT assay with titrated doses of M285–293 peptide. The data shown are representative of three independent experiments.

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We have identified the peptide from the M protein that is recognized by SV5-specific cells. Thus, to confirm our data obtained with CD8 blocking, functional avidity was measured by testing the peptide requirement of M-specific CD8+ T cells. CD8+ T cells isolated from the MLN of d3 or d7 mice were tested for their ability to respond to titrated concentrations of the immunodominant, Ld-restricted M285–293 peptide. At d7, the amount of peptide required to elicit the 50% maximal response was ∼1000-fold more than that required by cells at d3 (Fig. 2 B). These data confirm the observation that at d3, high avidity cells dominate the response, but at later time points, low avidity cells emerge.

At d3, the M-specific response was equivalent in size to the other SV5-specific responses (Fig. 2,A). However, by d5, a clear immunodominance pattern was present, with the M-specific response clearly established as immunodominant. The same avidity and immunodominance phenotypes detected at d5 continued into the memory population (d40) (Fig. 2 A).

In contrast to the MLN, in the spleen the M-specific response was the largest response at every time point tested, even d5, which was the earliest time at which virus-specific cells could be detected (Fig. 3). Although the responses at d5 were small in three separate experiments performed, the M-specific response was the largest response in each experiment and was statistically different in two of the three experiments (p ≤ 0.05). In addition, the d5 population contained both high and low avidity cells (Fig. 3). The immunodominance and avidity patterns, as well as the kinetic delay, suggest that the CD8+ T cells detected in the spleen were activated in the MLN, entered circulation, and subsequently entered the spleen.

FIGURE 3.

Delayed kinetics in the detection of SV5-specific CD8+ T cells in the spleen. BALB/c mice were immunized i.n. with 1 × 106 PFU of WT rSV5, and splenocytes were isolated and pooled from four mice for each time point. ELISPOT assays were performed in the absence (hatched bars) or presence (filled bars) of anti-CD8-blocking Ab using P815 cells, as described in Fig. 2 A, to stimulate production of IFN-γ. The data shown are representative of three independent experiments.

FIGURE 3.

Delayed kinetics in the detection of SV5-specific CD8+ T cells in the spleen. BALB/c mice were immunized i.n. with 1 × 106 PFU of WT rSV5, and splenocytes were isolated and pooled from four mice for each time point. ELISPOT assays were performed in the absence (hatched bars) or presence (filled bars) of anti-CD8-blocking Ab using P815 cells, as described in Fig. 2 A, to stimulate production of IFN-γ. The data shown are representative of three independent experiments.

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Together these findings indicate that following i.n. immunization with SV5, high avidity CD8+ T cells initially respond (d3) to the infection. However, by d5 postinfection, low avidity cells are recruited into the response. In addition, immunodominance does not emerge at early times postinfection (d3), but becomes evident as the SV5-specific response progresses. Thus, the characteristics of the T cell population present at the height of the antiviral response to SV5 infection do not reflect the initial response. These data provide evidence that there is a maturation of the immune response following SV5 infection. Although the emergence of low avidity CD8+ T cells and immunodominance are kinetically linked, whether they are causally linked is unknown.

Although the presence of T cells in the lung following infection with a number of viruses has been reported, the avidity of the cells present was not assessed (16, 23, 24). Given the critical nature of avidity in determining clearance efficacy in other viral models (10, 11, 12), it was important to determine the avidity of SV5-specific CD8+ T cells present at the site of infection. At various time points postinfection with SV5, cells were isolated from the lung and analyzed directly ex vivo by ELISPOT assay. As previously noted, the emergence of CD8+ T cells in the lung is delayed compared with the MLN (d3) with only very low numbers of detectable Ag-specific CD8+ effector T cells before d7 (Fig. 1). When the functional avidity was assessed, a striking observation was made. When compared with the response in the MLN, there was a major skewing toward low avidity cells in the lung at d9-d40 (Fig. 4). Interestingly, the responses to the subdominant P and HN epitopes were also underrepresented in the lung compared with the MLN.

FIGURE 4.

High avidity cells are highly underrepresented in the lung compared with the MLN and the spleen. BALB/c mice were immunized i.n. with 1 × 106 PFU of WT rSV5, and lymphocytes from the lungs of infected mice were isolated and pooled from four mice for each time point. ELISPOT assays were performed in the absence (hatched bars) or presence (filled bars) of anti-CD8-blocking Ab using P815 cells, as described in Fig. 2 A, to stimulate production of IFN-γ. The data shown are representative of three independent experiments.

FIGURE 4.

High avidity cells are highly underrepresented in the lung compared with the MLN and the spleen. BALB/c mice were immunized i.n. with 1 × 106 PFU of WT rSV5, and lymphocytes from the lungs of infected mice were isolated and pooled from four mice for each time point. ELISPOT assays were performed in the absence (hatched bars) or presence (filled bars) of anti-CD8-blocking Ab using P815 cells, as described in Fig. 2 A, to stimulate production of IFN-γ. The data shown are representative of three independent experiments.

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SV5 is known to replicate in the lungs of infected mice (25). In addition, infection of cells in vitro shows that SV5 can replicate to high titers without producing apparent cytopathic effects. Conversely, vaccinia virus infection is associated with cytopathic effects and has been shown to spread systemically following i.n. infection in mice (26). To determine whether the early emergence (d3) of high avidity T cells was specific to SV5 infection, BALB/c mice were immunized i.n. with either a recombinant vaccinia virus expressing the full-length P protein (rVV-P) or M protein (rVV-M) from SV5. At d3 postinfection, responder cells from immunized mice were harvested and analyzed by ELISPOT. Similar to SV5 infection, both rVV-P and rVV-M infection elicited almost exclusively high avidity CD8+ T cells in the MLN (Fig. 5). In contrast to SV5 infection, however, the responses in the spleen of vaccinia virus-immunized mice were also exclusively high avidity at d3 (Fig. 5). This finding is most likely the result of the systemic spread of the virus, resulting in the activation of T cells in the spleen. Similar to SV5, at d5 postvaccinia virus infection, low avidity M-specific CD8+ T cells began to emerge in both the MLN and the spleen, although there was a lower percentage of M-specific low avidity cells detected at d5 as a result of vaccinia virus infection compared with SV5 infection (64.2 ± 4.2% with SV5 infection compared with 30.5 ± 6.4% with vaccinia virus infection). Consistent with our previous results (3), the P-specific response remained exclusively high avidity (Fig. 5). The percentage of M-specific cells that were low avidity continued to increase through d7. Interestingly, there was a larger proportion of low avidity M-specific CD8+ T cells detected in the spleen compared with the MLN, suggesting that there may be a skewing toward the elicitation of high avidity cells in the draining lymph node following vaccinia virus infection. In summary, respiratory infection with either SV5 or vaccinia virus resulted in an initial population of cells (d3) that was almost exclusively high avidity. Whether this is the case for other virus infections is currently under investigation. In addition, these findings demonstrate that the high avidity response elicited at early times postinfection is not exclusive to the draining lymph node, but may also occur in other lymphoid tissues, such as the spleen.

FIGURE 5.

The high avidity CTL response at early times (d3) postinfection is a general property of the antiviral response. Mice were immunized i.n. with 104 PFU of either rVV-P or rVV-M, and ELISPOT assays were performed on cells isolated from the MLN and the spleen. Cells were cocultured with P815 stimulators infected with SV5 in the absence (hatched bars) or presence (filled bars) of anti-CD8-blocking Ab. As a negative control, responder cells were plated together with uninfected P815 cells in the absence (cross-hatched bars) and presence (gray bars) of anti-CD8-blocking Ab. The data shown are representative of three independent experiments.

FIGURE 5.

The high avidity CTL response at early times (d3) postinfection is a general property of the antiviral response. Mice were immunized i.n. with 104 PFU of either rVV-P or rVV-M, and ELISPOT assays were performed on cells isolated from the MLN and the spleen. Cells were cocultured with P815 stimulators infected with SV5 in the absence (hatched bars) or presence (filled bars) of anti-CD8-blocking Ab. As a negative control, responder cells were plated together with uninfected P815 cells in the absence (cross-hatched bars) and presence (gray bars) of anti-CD8-blocking Ab. The data shown are representative of three independent experiments.

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There is evidence that the responding CD8+ T cell population following initial exposure to Ag is often different from the cells that are elicited following subsequent exposures to the same Ag. For example, it has been shown that following secondary exposure there is a decrease in the diversity of TCR Vβ segments used in the responding CD8+ T cell population (27). In addition, following secondary influenza virus infection, the relative dominance of some T cell epitopes is diminished when compared with the primary response (28, 29). Thus, it was of interest to determine whether the observed pattern of immunodominance and, more importantly, the early emergence of high avidity cells were specific to the primary immune response. Mice were initially immunized i.n. with 1 × 106 PFU of WT SV5. At d40 postinfection, mice were challenged i.n. with 1 × 107 PFU of the same virus. Responders were harvested and analyzed by ELISPOT d2 after secondary immunization. Day two was the earliest time at which Ag-specific expansion could be detected (data not shown). As a control, mice that were initially infected with SV5 were challenged with PBS and analyzed at d2. There was a significant expansion (∼20-fold) of Ag-specific CD8+ T cells in the MLN d2 after challenge with SV5 compared with the negative control (Fig. 6,A). As was observed in the primary response, the earliest detectable responding SV5-specific cells were exclusively high avidity. Interestingly, immunodominance was apparent in the secondary response even at d2, as evidenced by the larger response to the M epitope compared with P, F, and HN (Fig. 6 A). Thus, once established following primary SV5 exposure, immunodominance is a constant feature of the SV5-specific response. The response in the spleen was not significantly different from the PBS-challenged mice, demonstrating a lag in the response in the spleen analogous to that observed during the primary response. Together, these data demonstrate that the early appearance of high avidity cells in the MLN is not specific to the primary response to virus, but rather is a general characteristic of the antiviral CD8+ T cell response to SV5.

FIGURE 6.

The emergence of high avidity CTL at the initiation of the immune response is independent of previous exposure to Ag. Mice were initially immunized i.n. with 106 PFU of WT rSV5. At d40 postinfection, mice were given 107 PFU of either WT rSV5 or PBS. At d2 (A), d4 (B), and d7 (C), postsecondary infection cells were isolated from the MLN and spleen. The cells from three mice were pooled. ELISPOT assays were performed in the absence (hatched bars) or presence (filled bars) of anti-CD8-blocking Ab using P815 cells, as described in Fig. 2 A, to stimulate production of IFN-γ. The animals receiving PBS were used to assess specific expansion as a result of secondary exposure. The data shown are representative of three independent experiments.

FIGURE 6.

The emergence of high avidity CTL at the initiation of the immune response is independent of previous exposure to Ag. Mice were initially immunized i.n. with 106 PFU of WT rSV5. At d40 postinfection, mice were given 107 PFU of either WT rSV5 or PBS. At d2 (A), d4 (B), and d7 (C), postsecondary infection cells were isolated from the MLN and spleen. The cells from three mice were pooled. ELISPOT assays were performed in the absence (hatched bars) or presence (filled bars) of anti-CD8-blocking Ab using P815 cells, as described in Fig. 2 A, to stimulate production of IFN-γ. The animals receiving PBS were used to assess specific expansion as a result of secondary exposure. The data shown are representative of three independent experiments.

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To determine whether the maturation of the secondary effector populations, i.e., the appearance of low avidity T cells, was similar to that seen in the primary response, ELISPOT assays were performed on responder cells isolated d4 and d7 postsecondary challenge. By d4 postchallenge, low avidity cells begin to emerge in the M-, F-, and HN-specific responses in the MLN, as was seen at d5 in the primary response (Fig. 6,B). There was little change in the splenic responding population at this time point. As the response in the MLN begins to decline by d7, the response in the spleen had increased significantly (Fig. 6,C). However, there was no change in the immunodominance hierarchies or the avidity of the SV5-specific responses compared with the response following primary exposure to SV5 (Fig. 2). Taken together, these results indicate that after the initial response to virus, subsequent exposures to the SV5 do not change the relative avidity of the responding population. In addition, once established in the primary response, immunodominance is present even at the earliest time at which virus-induced expansion of the CD8+ T cells can be detected following secondary exposure to SV5.

Numerous studies have implicated high avidity CD8+ T cells in the efficient clearance of virus in vivo (10, 11, 12, 30). To investigate the kinetics of the emergence of high and low avidity cells following virus infection of the respiratory tract, we used a modified ELISPOT assay that allows for the enumeration of high and low avidity T cells either by the inclusion of anti-CD8-blocking Ab or by peptide titration. We have defined an important feature of the antiviral CD8+ T cell responses using two unrelated viruses, SV5 and vaccinia virus. At early times after i.n. virus infection (d3), the first detectable responding cells are almost exclusively high avidity, with the subsequent emergence of low avidity cells (d5). In addition to a skewed avidity profile, we made the observation that immunodominance is absent at early times (d3) after viral infection. The lack of immunodominance at early times postinfection is in agreement with results of others (16, 31). Once established, however, it remains unchanged following secondary exposure to Ag.

The most interesting observation in our studies was that high avidity CD8+ T cells were selectively activated and/or expanded at early times following infection, as indicated by the response in the MLN at d3 (Fig. 2,A), which was almost exclusively high avidity cells. This was followed by the later emergence of low avidity cells as the response progressed. In contrast, the response in the spleen consisted of relatively low numbers of cells until d7 postinfection, at which time a mixture of high and low avidity CD8+ T cells was detected (Fig. 3). As i.n. infection with SV5 results in an infection in the respiratory tract (25), it is likely the cells that are detected in the spleen at d5 are cells that were initially activated in the MLN and then migrated to the spleen. This interpretation is supported by the results of a recent study that analyzed the response to cutaneous infection with HSV 1 (32). This immune response was initiated in the draining lymph node by d2 postinfection. However, CD8+ T cells were not detected in the spleen until d4 postinfection.

Our data with vaccinia virus infection provide further evidence for the selective activation and/or expansion of high avidity CD8+ T cells at the initiation of the response. As seen with SV5, i.n. infection of BALB/c mice with recombinant vaccinia viruses (rVV-P or rVV-M) resulted in exclusively high avidity P- and M-specific CD8+ effector T cells at d3 postinfection in the MLN and the spleen (Fig. 5). Intranasal infection of BALB/c mice with the WR strain of vaccinia causes a systemic infection with detectable levels of virus in the blood and the spleen by d3 postinfection (26). Thus, it is likely that naive cells are activated initially in the spleen as well as the MLN. An exclusively high avidity early antiviral response was also detected after a secondary challenge with SV5 (d2 postchallenge) (Fig. 6,A). In total, these results demonstrate that the high avidity nature of the initial responding CD8+ T cells is a property of both the primary and secondary antiviral response following respiratory tract infection with SV5 or vaccinia virus. These results differ from a study that showed a time-dependent increase in the avidity of cells specific for an epitope from lymphocytic choriomeningitis virus (15). The reason for this discrepancy in results is not known, but could reflect a difference in the route of infection or the virus used. In striking contrast to the responses detected in the spleen and the MLN, the effector population in the lung following infection with SV5 showed a dramatic skewing toward low avidity cells (Fig. 4). The mechanism responsible for the underrepresentation of high avidity cells is currently under investigation.

The mechanism responsible for the emergence of the disparate populations present at d3 vs d5 is not known. Certainly, the environment present at d1-d2, which is directing the populations detected at d3, could be significantly different from that present at later times. One factor that may change over the course of infection and that could influence the populations present is the level of peptide presentation.

Peptide/MHC density has been shown to play a crucial role in vitro in the generation of CTL of a defined avidity (10, 12, 30, 33). In these studies, low concentrations of peptide were found to preferentially expand high avidity CD8+ T cells, while high concentrations of peptide stimulated proliferation of low avidity CTL. It is currently not known whether peptide/MHC density exerts a similar pressure on the activation of naive precursors in vivo. However, based on the in vitro studies, it is attractive to hypothesize that high avidity precursors require less Ag than low avidity precursors to initially become activated. This model would predict that at early times postinfection, virus peptide/MHC determinant densities are low, and thus only sufficient to activate high avidity cells. However, over time, determinant densities may have reached sufficient levels to also activate low avidity cells, thus accounting for the lag in detection of low avidity cells in our experiments. Our data support this model and indicate that by d5 there is a >30-fold increase in the number of SV5-specific low avidity cells detected in the MLN compared with d3 postinfection. However, there is only a 5-fold increase in the number of high avidity cells. The mechanism by which peptide could be limited early on postinfection is unknown, but factors such as cytokine environment could explain enhanced levels of peptide at later times postinfection. For example, it is well established that IFN mediates up-regulation of proteins that facilitate Ag processing and presentation as well as immunoproteasome formation (34). It is possible that at early times postinfection, the composition of the proteosomes is such that only low levels of immunogenic epitopes are presented, promoting preferential activation of high avidity cells. However, during the course of a viral infection, infected cells are triggered to synthesize cytokines that create an inflammatory environment. These soluble mediators of inflammation have been shown to activate APCs, which allows for more efficient priming of Ag-specific T cells (35). Therefore, it is possible that once a potent inflammatory environment is established, low avidity cells are able to overcome any lack of stimulatory agents (i.e., limiting Ag dose and cytokines) that are not present at sufficient levels during the beginning of the infection, and thus are activated at later time points.

In summary, the study presented in this work provides new and important insights into the emergence and maturation of the antiviral response following respiratory tract infection with two different viruses. The results show that at d3 postinfection, there is no evidence of immunodominance. However, by d5, immunodominance hierarchies are permanently established. Furthermore, the initial CD8+ T cells present in these antiviral populations in the MLN are exclusively high avidity. The high avidity response at early times postinfection is independent of subsequent exposures to Ag and of the virus administered. Elucidation of the mechanisms responsible for the control of the selective activation and expansion of high avidity T cells and the emergence of immunodominance may allow for the design of vaccines that elicit high avidity cells to multiple epitopes, which should provide optimal protection against pathogens.

We thank Drs. Steven B. Mizel, Jason M. Grayson, and Ellen M. Palmer for critical review of the manuscript. We thank Robert Lamb and Reay Paterson for rVV-expressing HN and F.

1

This work was supported by National Institutes of Health Grants AI 43591 (to M.A.A.-M.) and AI 46282 (to G.D.P.). P.M.G. was supported by National Research Training Award Grant AI07401. In addition, this work was supported in part by Grant 02 819-30-RGV (to G.D.P.) from the American Foundation for AIDS Research (amfAR).

3

Abbreviations used in this paper: SV5, simian virus 5; d, day; i.n., intranasal; MLN, mediastinal lymph node; VV, vaccinia virus; WT, wild type.

1
Hou, S., P. C. Doherty, M. Zijlstra, R. Jaenisch, J. M. Katz.
1992
. Delayed clearance of Sendai virus in mice lacking class I MHC-restricted CD8+ T cells.
J. Immunol.
149
:
1319
2
Allan, W., Z. Tabi, A. Cleary, P. C. Doherty.
1990
. Cellular events in the lymph node and lung of mice with influenza: consequences of depleting CD4+ T cells.
J. Immunol.
144
:
3980
3
Gray, P. M., G. D. Parks, M. A. Alexander-Miller.
2001
. A novel CD8-independent high-avidity cytotoxic T-lymphocyte response directed against an epitope in the phosphoprotein of the paramyxovirus simian virus 5.
J. Virol.
75
:
10065
4
Yewdell, J. W., J. R. Bennink.
1999
. Immunodominance in major histocompatibility complex class I-restricted T lymphocyte responses.
Annu. Rev. Immunol.
17
:
51
5
Deng, Y., J. W. Yewdell, L. C. Eisenlohr, J. R. Bennink.
1997
. MHC affinity, peptide liberation, T cell repertoire, and immunodominance all contribute to the paucity of MHC class I-restricted peptides recognized by antiviral CTL.
J. Immunol.
158
:
1507
6
Wallace, M. E., R. Keating, W. R. Heath, F. R. Carbone.
1999
. The cytotoxic T-cell response to herpes simplex virus type 1 infection of C57BL/6 mice is almost entirely directed against a single immunodominant determinant.
J. Virol.
73
:
7619
7
Busch, D. H., E. G. Pamer.
1998
. MHC class I/peptide stability: implications for immunodominance, in vitro proliferation, and diversity of responding CTL.
J. Immunol.
160
:
4441
8
Sijts, A. J., M. L. De Bruijn, M. E. Ressing, J. D. Nieland, E. A. Mengede, C. J. Boog, F. Ossendorp, W. M. Kast, C. J. Melief.
1994
. Identification of an H-2 Kb-presented Moloney murine leukemia virus cytotoxic T-lymphocyte epitope that displays enhanced recognition in H-2Db mutant bm13 mice.
J. Virol.
68
:
6038
9
Gegin, C., F. Lehmann-Grube.
1992
. Control of acute infection with lymphocytic choriomeningitis virus in mice that cannot present an immunodominant viral cytotoxic T lymphocyte epitope.
J. Immunol.
149
:
3331
10
Alexander-Miller, M. A., G. R. Leggatt, J. A. Berzofsky.
1996
. Selective expansion of high- or low-avidity cytotoxic T lymphocytes and efficacy for adoptive immunotherapy.
Proc. Natl. Acad. Sci. USA
93
:
4102
11
Derby, M., M. Alexander-Miller, R. Tse, J. Berzofsky.
2001
. High-avidity CTL exploit two complementary mechanisms to provide better protection against viral infection than low-avidity CTL.
J. Immunol.
166
:
1690
12
Sedlik, C., G. Dadaglio, M. F. Saron, E. Deriaud, M. Rojas, S. I. Casal, C. Leclerc.
2000
. In vivo induction of a high-avidity, high-frequency cytotoxic T-lymphocyte response is associated with antiviral protective immunity.
J. Virol.
74
:
5769
13
Constant, S. L., K. S. Lee, K. Bottomly.
2000
. Site of antigen delivery can influence T cell priming: pulmonary environment promotes preferential Th2-type differentiation.
Eur. J. Immunol.
30
:
840
14
Alexander-Miller, M. A..
2000
. Differential expansion and survival of high and low avidity cytotoxic T cell populations during the immune response to a viral infection.
Cell. Immunol.
201
:
58
15
Slifka, M. K., J. L. Whitton.
2001
. Functional avidity maturation of CD8+ T cells without selection of higher affinity TCR.
Nat. Immun.
2
:
711
16
Belz, G. T., P. G. Stevenson, P. C. Doherty.
2000
. Contemporary analysis of MHC-related immunodominance hierarchies in the CD8+ T cell response to influenza A viruses.
J. Immunol.
165
:
2404
17
Marshall, D. R., S. J. Turner, G. T. Belz, S. Wingo, S. Andreansky, M. Y. Sangster, J. M. Riberdy, T. Liu, M. Tan, P. C. Doherty.
2001
. Measuring the diaspora for virus-specific CD8+ T cells.
Proc. Natl. Acad. Sci. USA
98
:
6313
18
Alexander, M. A., C. A. Damico, K. M. Wieties, T. H. Hansen, J. M. Connolly.
1991
. Correlation between CD8 dependency and determinant density using peptide-induced, Ld-restricted cytotoxic T lymphocytes.
J. Exp. Med.
173
:
849
19
Speiser, D. E., D. Kyburz, U. Stubi, H. Hengartner, R. M. Zinkernagel.
1992
. Discrepancy between in vitro measurable and in vivo virus neutralizing cytotoxic T cell reactivities: low T cell receptor specificity and avidity sufficient for in vitro proliferation or cytotoxicity to peptide-coated target cells but not for in vivo protection.
J. Immunol.
149
:
972
20
Kwan-Lim, G. E., T. Ong, F. Aosai, H. Stauss, R. Zamoyska.
1993
. Is CD8 dependence a true reflection of TCR affinity for antigen?.
Int. Immunol.
5
:
1219
21
Alexander-Miller, M. A., G. R. Leggatt, A. Sarin, J. A. Berzofsky.
1996
. Role of antigen, CD8, and cytotoxic T lymphocyte (CTL) avidity in high dose antigen induction of apoptosis of effector CTL.
J. Exp. Med.
184
:
485
22
Van Emmerik, N. E., C. R. Daane, C. J. Knoop, C. Hesse, L. M. Vaessen, A. H. Balk, B. Mochtar, F. H. Claas, W. Weimar.
1997
. The avidity of allospecific cytotoxic T lymphocytes (CTL) determines their cytokine production profile.
Clin. Exp. Immunol.
110
:
447
23
Chang, J., A. Srikiatkhachorn, T. J. Braciale.
2001
. Visualization and characterization of respiratory syncytial virus F-specific CD8+ T cells during experimental virus infection.
J. Immunol.
167
:
4254
24
Mo, X. Y., R. A. Tripp, M. Y. Sangster, P. C. Doherty.
1997
. The cytotoxic T-lymphocyte response to Sendai virus is unimpaired in the absence of γ interferon.
J. Virol.
71
:
1906
25
Young, D. F., R. E. Randall, J. A. Hoyle, B. E. Souberbielle.
1990
. Clearance of a persistent paramyxovirus infection is mediated by cellular immune responses but not by serum-neutralizing antibody.
J. Virol.
64
:
5403
26
Smee, D. F., K. W. Bailey, M. H. Wong, R. W. Sidwell.
2001
. Effects of cidofovir on the pathogenesis of a lethal vaccinia virus respiratory infection in mice.
Antiviral Res.
52
:
55
27
Busch, D. H., I. Pilip, E. G. Pamer.
1998
. Evolution of a complex T cell receptor repertoire during primary and recall bacterial infection.
J. Exp. Med.
188
:
61
28
Belz, G. T., W. Xie, J. D. Altman, P. C. Doherty.
2000
. A previously unrecognized H-2Db-restricted peptide prominent in the primary influenza A virus-specific CD8+ T-cell response is much less apparent following secondary challenge.
J. Virol.
74
:
3486
29
Belz, G. T., W. Xie, P. C. Doherty.
2001
. Diversity of epitope and cytokine profiles for primary and secondary influenza A virus-specific CD8+ T cell responses.
J. Immunol.
166
:
4627
30
Zeh, H. J., III, D. Perry-Lalley, M. E. Dudley, S. A. Rosenberg, and J. C. Yang. 1999. High avidity CTLs for two self-antigens demonstrate superior in vitro and in vivo antitumor efficacy. J. Immunol. 162:989.
31
Chen, W., L. C. Anton, J. R. Bennink, J. W. Yewdell.
2000
. Dissecting the multifactorial causes of immunodominance in class I-restricted T cell responses to viruses.
Immunity
12
:
83
32
Coles, R. M., S. N. Mueller, W. R. Heath, F. R. Carbone, A. G. Brooks.
2002
. Progression of armed CTL from draining lymph node to spleen shortly after localized infection with herpes simplex virus 1.
J. Immunol.
168
:
834
33
Pihlgren, M., P. M. Dubois, M. Tomkowiak, T. Sjogren, J. Marvel.
1996
. Resting memory CD8+ T cells are hyperreactive to antigenic challenge in vitro.
J. Exp. Med.
184
:
2141
34
Chen, W., C. C. Norbury, Y. Cho, J. W. Yewdell, J. R. Bennink.
2001
. Immunoproteasomes shape immunodominance hierarchies of antiviral CD8+ T cells at the levels of T cell repertoire and presentation of viral antigens.
J. Exp. Med.
193
:
1319
35
Banchereau, J., F. Briere, C. Caux, J. Davoust, S. Lebecque, Y. J. Liu, B. Pulendran, K. Palucka.
2000
. Immunobiology of dendritic cells.
Annu. Rev. Immunol.
18
:
767