| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Receptor in Immune Regulation1
Department of Pathobiological Sciences, University of Wisconsin, Madison, WI 53706
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
R deficiency altered the epitope hierarchy of the pool of LCMV-specific memory CD8 T cells without significantly affecting the immunodominance of the primary CD8 T cell response in an acute infection. Taken together, these findings should further our understanding about the regulation of T cell responses in human chronic viral infections. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
and TNF-
. CD8 T cells recognize peptidic Ags derived from intracellular proteins, complexed with cell surface MHC class I molecules. Typically, polyclonal CD8 T cell responses are directed against multiple epitopes, and the number of CD8 T cells responding to each epitope varies depending on the immunodominance hierarchy (11). The epitopes that elicit strong CD8 T cell responses are referred to as immunodominant epitopes, and those epitopes that stimulate weak CTL responses are termed subdominant epitopes (11). Studies on immunodominance hierarchy during acute viral infections have shown that the epitope hierarchy established during the primary response is remarkably well preserved in the pool of the memory T cells (12, 13). However, the effect of a chronic viral infection on the epitope hierarchy, function, and phenotype of virus-specific memory CD8 T cells is not well characterized. To address these issues, there is a need to compare the responses of CD8 T cells to a given set of epitopes under conditions of acute and chronic viral infections. The LCMV system allows us to examine CD8 T cell responses to multiple epitopes using two viral strains in which the CTL epitopes are identical but differ in the duration of infection in the host (13, 14, 15, 16, 17). In this study, we compared the regulation of CD8 T cell responses to dominant and subdominant epitopes between an acute and a chronic LCMV infection in C57BL/6 mice. Specifically, we asked the following questions: 1) are there differences in immunodominance in the primary and memory CD8 T cell responses between an acute and a chronic LCMV infection, 2) what is the effect of a chronic LCMV infection on the ability of memory CD8 T cells to produce cytokines upon antigenic stimulation in vitro, 3) what is the effect of chronic LCMV infection on the expression of L-selectin (CD62L), CD44, and CCR7 on virus-specific memory CD8 T cells, and 4) what is the role of IFN-/IFN-R interactions in regulating immunodominance of the CD8 T cell response to LCMV? |
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
C57BL/6 mice were purchased from the National Cancer Institute (Bethesda, MD). IFN-R-deficient (IFN-R-/-) mice (18) were purchased from The Jackson Laboratory (Bar Harbor, ME). Mice were maintained under specific pathogen-free conditions in isolation cages with filter covers and were used at 6–8 wk of age in all experiments. All animal experiments were performed in strict accordance with the approved institutional animal welfare guidelines.
Virus and virus titration
The Armstrong (LCMV-Arm) and T1B (LCMV-T1b) strains of LCMV were used in this study (16, 17). To induce an acute infection, mice were injected i.p. with 2 x 105 PFU of LCMV-Arm. In some experiments, an acute infection was induced by i.v. administration of 2 x 104 or 2 x 105 or 2 x 106 PFU of LCMV-Arm. A chronic infection was induced by injecting mice with 2 x 106 PFU of LCMV-T1b i.v. Infectious LCMV in the tissues and serum of infected mice was quantitated by plaque assay using Vero cell monolayers (17).
CTL assay
Primary ex vivo MHC class I (MHC-I)-restricted LCMV-specific cytotoxic activity in the spleens of LCMV-infected mice was measured by 51Cr-release assay using MC57 cells as target cells (17).
Flow cytometry
LCMV-specific CD8 T cells were visualized using MHC-I tetramers. The development and use of MHC-I tetramers complexed with the LCMV CTL epitope peptides NP396-404 (NP396), GP33-41 (GP33), and GP276-286 (GP276) in the viral nucleoprotein or glycoprotein has been described elsewhere (13). Freshly explanted spleen cells were stained with PerCP-labeled anti-CD8, PE-labeled anti-CD62L, FITC-labeled anti-CD44 Abs, and allophycocyanin-labeled MHC-I tetramers for 1 h at 4°C in FACS buffer (PBS containing 2% BSA and 0.1% sodium azide). After staining, cells were fixed in 2% paraformaldehyde and acquired on a FACSCalibur flow cytometer (BD Biosciences, Mountain View, CA). Flow cytometry data were analyzed using CellQuest software (BD Biosciences). Abs were purchased from BD PharMingen (San Diego, CA).
Intracellular staining for IFN-, Bcl-2, Bcl-3, and Bcl-6
Splenocytes were stimulated with various LCMV CTL epitope peptides for 5 h in the presence of brefeldin A (13). After culture, cells were stained for cell surface CD8, intracellular IFN-, and Bcl-2, Bcl-3, or Bcl-6 using the Cytofix/Cytoperm kit (BD PharMingen) (19). Abs to murine Bcl-2 and IFN- were purchased from BD PharMingen. Anti-Bcl-3 and anti-Bcl-6 Abs were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
Quantitation of CCR7 expression on LCMV-specific CD8 T cells using CCL19-Fc fusion proteins
CCL19-Fc fusion proteins were kindly provided by J. Cyster (University of California, San Francisco) (20). Splenocytes were first incubated with anti-mouse CD16/CD32 to block Fc receptors and then were stained with CCL19-Fc, PerCP-conjugated anti-CD8, FITC-conjugated anti-CD62L, and allophycocyanin-labeled MHC-I tetramers. CCL19-Fc binding was visualized by a second step staining with PE-labeled anti-human Fc (Jackson ImmunoResearch Laboratories, West Grove, PA). As controls for binding specificity of CCL19-Fc, cells were preincubated with recombinant soluble chemokine CCL19 (macrophage-inflammatory protein-3
; R&D Systems, Minneapolis, MN) at a concentration of 5 µg/ml. Stained cells were fixed in 2% paraformaldehyde and acquired on a FACSCalibur flow cytometer (BD Biosciences), and data were analyzed as described above.
Statistical analysis
Experimental data were analyzed using commercially available statistical software (SYSTAT, version 8.0; Chicago, IL). Groups were compared by Student’s t test and significance was defined at p 
0.05.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Infection of immunocompetent mice with the Armstrong strain of LCMV results in an acute infection, which is resolved in 8–10 days (13, 17). In striking contrast, infection of mice with T1b strain of LCMV causes a chronic infection, lasting up to 30 days (16, 21). Early in the infection, both LCMV-Arm and LCMV-T1b viruses replicate predominantly in the tissue macrophages found in spleen, lymph nodes, liver, lung, etc. (22). In LCMV-Arm-infected mice, after attaining peak viral titers on day 3 postinfection (PI), viral load in the tissues drops precipitously after day 5 PI and most of the infection is resolved by day 8 PI (21). LCMV-Arm replication is restricted to tissue macrophages with very little spread to the parenchymal cells (22). Unlike LCMV-Arm, which is rapidly controlled, LCMV-T1b spreads rapidly from macrophages to parenchymal cells, resulting in widespread dissemination to most visceral organs (22). Despite being biologically different, LCMV-Arm and LCMV-T1b are >99.8% genetically identical and the amino acid changes are not found in the MHC-I-restricted CTL epitopes (23). On day 8 PI, we compared MHC-I-restricted cytotoxic activity in the spleen between LCMV-Arm- and LCMV-T1b-infected mice. Consistent with published findings (17, 21), the cytotoxic activity in the spleens of LCMV-T1b-infected mice was substantially lower compared with LCMV-Arm-infected mice (data not shown). As expected, by day 8 PI, all of the LCMV-Arm-infected mice had resolved the infection and no infectious virus was detected in the liver and lungs. On day 8 PI, the viral titers in the liver and lungs of LCMV-T1b-infected mice were 6.5 ± 0.2 and 6.8 ± 0.2 (log10 PFU/gram of tissue), respectively. Next, we used MHC-I tetramers to compare the effect of chronic infection with LCMV-T1b on the expansion of CD8 T cells specific to the two Db-restricted immunodominant epitopes NP396 and GP33. As shown in Fig. 1A, high numbers of LCMV-specific CD8 T cells were detected in the spleens of LCMV-Arm-infected mice. The percentages of NP396- and GP33-specific CD8 T cells in LCMV-T1b-infected mice were significantly reduced, in comparison with LCMV-Arm-infected mice. It is worth emphasizing that a previous study had examined CD8 T cell responses to the immunodominant epitopes only during an LCMV-T1b infection between wild-type and CD4-deficient mice (16). However, this past study did not compare primary CD8 T cell responses with the immunodominant epitopes between LCMV-Arm and LCMV-T1b (chronic) in wild-type mice (16). The present study shows that a chronic LCMV infection reduced the expansion (clonal burst size) of CD8 T cells specific to the two immunodominant LCMV CTL epitopes, as compared with an acute LCMV infection.
|
; Fig. 1B) because staining with MHC-I tetramers yielded similar results (Fig. 1A). It is noteworthy that the epitope GP33 is presented by both Db and Kb MHC-I molecules (24). Whereas MHC-I tetramers bind only Db-restricted GP33-specific CD8 T cells, intracellular cytokine staining detects both Db- and Kb-restricted GP33-specific CD8 T cells (24). Therefore, percentages of GP33-specific IFN--producing CD8 T cells are more than MHC-I tetramer-binding CD8 T cells (compare Fig. 1, A and B). The total number of CD8 T cells specific to various epitopes in LCMV-Arm- and LCMV-T1b-infected mice is shown in Fig. 1C. In LCMV-T1b-infected mice, the total numbers of CD8 T cells specific to the epitopes NP396, GP33, and GP276 were reduced by 5.4-, 2.4-, and 4-fold, respectively, as compared with LCMV-Arm-infected mice (Fig. 1C). Interestingly, the expansion of NP205- and GP118-specific CD8 T cells was reduced by only 
1.6-fold in LCMV-T1b-infected mice. The epitope hierarchy of the primary CD8 T cell response in LCMV-Arm-infected mice was GP33 
NP396 > GP276 > NP205 GP118 (Fig. 1, B and C). In LCMV-T1b-infected mice, despite the high antigenic load, the epitope hierarchy of the primary CD8 T cell response was only slightly altered: although the most dominant epitope was GP33, which was followed by NP396, epitopes GP276, NP205, and GP118 were equally represented (Fig. 1, B and C). In summary, these data suggested that the antigenic load and duration of stimulation are critical factors that can affect the expansion of LCMV-specific CD8 T cells during the primary response.
Previous studies have shown that the number of memory CD8 T cells generated is determined by the magnitude of expansion during the primary response (original clonal burst size) to an acute viral infection (13). Accordingly, the epitope hierarchy established in the primary CD8 T cell response is conserved in the pool of memory T cells (13). However, the relationship between epitope hierarchy of the primary T cell response and the memory T cell pool after a chronic viral infection is not well understood. Therefore, we investigated the effect of chronic LCMV infection on epitope composition of CD8 T cell memory. One hundred twenty days after infection with LCMV-Arm or LCMV-T1b, the number of LCMV-specific memory CD8 T cells was determined by intracellular cytokine staining. At this time point, no infectious LCMV was detected in the lungs, liver, and brain of LCMV-Arm- and LCMV-T1b-infected mice (data not shown). LCMV-Arm and LCMV-T1b infection should have been resolved by approximately 10 and 30 days PI, respectively (16, 21). As shown in Fig. 2, A and B, memory CD8 T cells specific to all of the LCMV CTL epitopes were readily detected in both LCMV-Arm- and LCMV-T1b-infected mice. Fig. 2a illustrates the striking differences in the epitope hierarchy of LCMV-specific CD8 T cells between LCMV-Arm- and LCMV-T1b-infected mice. In LCMV-Arm-infected mice, the epitope hierarchy was as follows: GP33 > NP396 > GP276 > NP205 GP118. In striking contrast, the epitope hierarchy of LCMV-specific memory T cells in LCMV-T1b-infected mice was GP33 GP276 > NP396 GP118 > NP205. Most notably, epitope GP276, which was subdominant in the acute infection with LCMV-Arm, emerged as a dominant epitope after a chronic infection with LCMV-T1b. Furthermore, GP118 had become clearly dominant over NP205 in LCMV-T1b-infected mice. To more clearly define epitope hierarchy, we calculated the relative proportions of epitope-specific CD8 T cells among total LCMV-specific CD8 T cells during the primary and memory T cell responses. Data in Fig. 2C show the relative proportions of epitope-specific T cells of total LCMV-specific CD8 T cells on day 8 PI (primary) and day 120 PI (memory) in LCMV-Arm- and LCMV-T1b-infected mice. Consistent with published findings, data in Fig. 2C clearly show that, during an acute infection, the epitope hierarchy of LCMV-specific CD8 T cells in the primary response was conserved in the pool of memory T cells (13). Strikingly, in LCMV-T1b-infected mice, the epitope hierarchy of the LCMV-specific memory T cells was drastically different from the primary CD8 T cell response (Fig. 2C). In the memory phase, note that GP276 is codominant with GP33 in LCMV-T1b-infected mice (Fig. 2C). After an acute LCMV infection, the memory CD8 T cell pool represents a "scaled down" version of the primary response; irrespective of epitope specificity, >95% of the LCMV-specific CD8 T cells are lost after day 8 PI (Figs. 1C and 2B). However, during a chronic LCMV infection, the magnitude of CD8 T cell contraction after day 8 PI seems to be epitope dependent (Figs. 1C and 2B). In LCMV-T1b-infected mice, 90–95% of CD8 T cells specific to NP396, GP33, GP118, and NP205 epitopes were lost after day 8 PI (Figs. 1C and 2B). However, remarkably only 70% of GP276-specific CD8 T cells were lost after day 8 PI in LCMV-T1b-infected mice (Figs. 1C and 2B). It is noteworthy that, in the above-described experiments, mice were infected with different doses of LCMV-Arm and LCMV-T1b (2 x 105 and 2 x 106 PFU of LCMV-Arm and LCMV-T1b, respectively). We questioned whether infection of mice with a "low dose" (2 x 105 PFU) of LCMV-T1b would affect the immunodominance hierarchy among LCMV-specific CD8 T cells during the primary and memory phases of the T cell response. Consistent with published data (25), despite being infected with a lower dose, LCMV-T1b established a chronic infection (data not shown). Similar to data shown in Figs. 1 and 2, this "low-dose" LCMV-T1b infection also led to alteration in the epitope hierarchy among LCMV-specific CD8 T cells in the memory pool, as compared with the primary response (data not shown).
|
, irrespective of epitope specificity. The mean fluorescence intensity of staining for IFN- in CD8 T cells from LCMV-Arm-infected mice was generally above 200. In stark contrast, memory CD8 T cells in LCMV-T1b-infected mice produced significantly lower amounts of IFN-, and the mean fluorescence intensity of staining for IFN- ranged between 95 and 153. CD8 T cells from LCMV-T1b-infected mice also produced lower amounts of TNF-, as compared with CD8 T cells from LCMV-Arm-infected mice (data not shown). It is worth emphasizing that infectious LCMV-T1b was below detection levels at the time of this analysis for IFN- production. Therefore, the observed reduction in IFN- production by CD8 T cells from LCMV-T1b-infected mice is distinct from the "stunned" phenotype (functional unresponsiveness) of CD8 T cells, which is associated with high viral load (16, 26).
To further characterize cytokine production, we determined the activation threshold of memory CD8 T cells in LCMV-Arm- and LCMV-T1b-infected mice by measuring IFN- production as a function of peptide concentration (day 150 PI). As shown in Fig. 3, LCMV-specific CD8 T cells from both LCMV-Arm- and LCMV-T1b-infected mice were exquisitely sensitive to antigenic stimulation, and the number of IFN--producing cells varied in a dose-dependent manner. The activation threshold of memory CD8 T cells from LCMV-T1b-infected mice was similar to those of LCMV-Arm-infected mice (Fig. 3). These data showed that alterations in epitope hierarchy in LCMV-T1b-infected mice were not associated with any detectable changes in the sensitivities of LCMV-specific memory CD8 T cells to antigenic stimulation in vitro.
|
Elegant studies have shown that survival of memory T cells might be dependent upon the induction of antiapoptotic molecules like Bcl-2, Bcl-3, and Bcl-6 (19, 27, 28). It was of interest to determine whether epitope-specific differences in the magnitude of CD8 T cell contraction during a chronic LCMV infection were related to the expression patterns of Bcl-2, Bcl-3, and Bcl-6 molecules. The expression of Bcl-2, Bcl-3, and Bcl-6 in LCMV-specific CD8 T cells was determined on day 8 PI, a time point that precedes the onset of the contraction phase of the CD8 T cell response. As shown in Fig. 4A, interestingly, the levels of Bcl-2 expression in LCMV-specific CD8 T cells in LCMV-T1b-infected mice were consistently higher than in LCMV-Arm-infected mice. However, Bcl-2 expression in CD8 T cells did not significantly differ between epitopes in either LCMV-Arm- or LCMV-T1b-infected mice. It is worth pointing out that Bcl-2 levels in activated LCMV-specific CD8 T cells in both LCMV-Arm- and LCMV-T1b-infected mice were lower than in CD8 T cells from uninfected naive mice (Fig. 4C). Fig. 4B illustrates that Bcl-3 was readily detected in LCMV-specific CD8 T cells from both LCMV-Arm- and LCMV-T1b-infected mice. However, the levels of Bcl-3 in virus-specific CD8 T cells were similar between LCMV-Arm- and LCMV-T1b-infected mice. Additionally, epitope specificity of CD8 T cells did not affect the expression levels of Bcl-3 in LCMV-Arm- or LCMV-T1b-infected mice. It is noteworthy that, unlike Bcl-2, Bcl-3 levels in CD8 T cells from uninfected naive mice and activated LCMV-specific CD8 T cells were comparable (Fig. 4C). The expression of Bcl-6 was below the level of detection in LCMV-specific CD8 T cells (data not shown). In summary, the expression levels of Bcl-2 and Bcl-3 were not affected by antigenic specificity in LCMV-Arm- or LCMV-T1b-infected mice.
|
We next looked for phenotypic differences in LCMV-specific memory CD8 T cells between LCMV-Arm- and LCMV-T1b-infected mice. Previous studies have shown that short-term TCR signaling induces rapid loss of CD62L expression followed by re-expression within 24–48 h (29). However, extended TCR stimulation might lead to transcriptional silencing of the CD62L locus and reduced expression of cell surface CD62L for long periods (29). On day 8 PI, at the peak of the T cell response, all of the LCMV-specific CD8 T cells in both LCMV-Arm- and LCMV-T1b-infected mice expressed greatly reduced levels of cell surface CD62L, as compared with naive T cells (data not shown). However, differences were notable in the proportions of CD62L-expressing memory CD8 T cells between LCMV-Arm- and LCMV-T1b-infected mice (Fig. 5A). Whereas 60–70% of LCMV-specific memory CD8 T cells in LCMV-Arm-infected mice expressed CD62L, only 20–40% of memory CD8 T cells were CD62L+ in LCMV-T1b-infected mice. These data suggested that CD62L expression on activated CD8 T cells might be regulated by duration of antigenic stimulation in vivo. However, it is worth noting that LCMV-specific memory CD8 T cells in both LCMV-Arm- and LCMV-T1b-infected mice were uniformly CD44high (Fig. 5A).
|
60% of LCMV-specific memory CD8 T cells in LCMV-Arm-infected mice. The proportion of CCR7 expressing LCMV-specific memory CD8 T cells was slightly lower (ranged from 40–50%) in LCMV-T1b-infected mice, as compared with LCMV-Arm-infected mice. Effect of viral dose on epitope hierarchy of CD8 T cell responses to LCMV
Next, we investigated the effect of viral dose on epitope hierarchy of the primary and memory pool of virus-specific CD8 T cells by infecting C57BL/6 mice with three different doses of LCMV-Arm by i.v. injection: 2 x 104 PFU (low dose), 2 x 105 PFU (medium dose), or 2 x 106 PFU (high dose). As shown in Fig. 6, in mice infected with a low or medium dose of LCMV-Arm, the epitope hierarchy of the primary and memory CD8 T cell pool was GP33 NP396 > GP276. In striking contrast, in high-dose-infected mice, the epitope hierarchy of the primary and memory CD8 T cell pool was different (Fig. 6). In high-dose LCMV-Arm-infected mice, the epitope hierarchy of the primary CD8 T cell response was GP33 NP396 > GP276 (Fig. 6), but during the memory phase it changed to GP33 > GP276 > NP396 (Fig. 6). Furthermore, although not dramatic, it is worth noting that the percentages of GP276-specific memory CD8 T cells in the spleen increased in a virus dose-dependent fashion. These data suggested that the viral dose might play an important role in the emergence of GP276 as a codominant epitope during an acute viral infection.
|
/IFN-R interactions in regulating epitope hierarchy during acute and chronic LCMV infection
Several steps in the pathway of Ag processing and presentation are sensitive to alterations by effects of cytokines like IFN- and TNF- (11). IFN- and TNF- affect Ag processing and presentation by up-regulating the expression of molecules like TAP, MHC-I, and proteosomal subunits (11). Loss of IFN- has been shown to alter the immunodominance of the primary CD8 T cell response to Listeria monocytogenes (LM) in mice (32). Furthermore, DNA immunization-induced alterations in immunodominance during an acute LCMV infection are regulated by IFN- (33). In this study, we investigated the role of IFN-/IFN-R interactions in regulating the epitope hierarchy of the CD8 T cell responses during an acute or a chronic LCMV infection. To determine the role of IFN- in regulating the CD8 T cell responses to an acute viral infection, groups of wild-type C57BL/6 (+/+) and IFN-R-/- mice were infected with LCMV-Arm. On day 8 PI, primary LCMV-specific CD8 T cell responses were analyzed in the spleen using intracellular staining for IFN-. As expected, LCMV-Arm-infected +/+ mice exhibited potent CD8 T cell responses. The epitope hierarchy of the anti-LCMV CD8 T cell response in +/+ mice was GP33 NP396 > GP276 > NP205 GP118 (Fig. 7B), which was identical with the data shown in Fig. 1B. Upon LCMV-Arm infection, IFN-R-/- mice mounted robust CD8 T cell responses directed against both dominant and subdominant LCMV CTL epitopes (Fig. 7, A and B). IFN- receptor deficiency had minimal effects on the epitope hierarchy of the primary anti-LCMV CD8 T cell response. The immunodominance hierarchy of the primary LCMV-specific CD8 T cell response in IFN-R-/- mice was NP396 GP33 > GP276 > GP118 > NP205 (Fig. 7). However, it is noteworthy that the expansion of LCMV-specific CD8 T cells (primary response) was significantly impaired in IFN-R-/- mice, as compared with +/+ mice (Fig. 7B). To examine the importance of IFN- in regulation of immunodominance hierarchy among the pool of LCMV-specific memory CD8 T cells, +/+ and IFN-R-/- mice were infected with LCMV-Arm, and CD8 T cell responses were analyzed 40 days later. As expected, the immunodominance hierarchy among LCMV-specific memory CD8 T cells in +/+ mice was GP33 > NP396 > GP276 > NP205 GP118 (Fig. 7B), which was the same as the primary response. On day 40 PI, memory CD8 T cells specific to all of the LCMV CTL epitopes were readily detected in the spleens of IFN-R-/- mice (Fig. 7A). As shown in Fig. 7, A and B, strikingly, in IFN-R-/- mice the epitope hierarchy of the memory CD8 T cells (day 40 PI) was different from that of the primary CD8 T cell response (day 8 PI). The immunodominance hierarchy among LCMV-specific memory CD8 T cells in IFN-R-/- mice was GP33 > NP396 GP276 > GP118 > NP205 (Fig. 7, A and B). In contrast with the primary response, GP33 is clearly dominant over all of the epitopes and, remarkably, GP276 and NP396 have become "equidominant" in the memory T cell compartment of IFN-R-/- mice. In summary, these data suggested that IFN-R deficiency could lead to alterations in the epitope hierarchy in the pool of LCMV-specific memory CD8 T cells with little effect on the immunodominance during the primary response.
|
R deficiency on the epitope hierarchy of the CD8 T cell response during a chronic LCMV infection. Groups of +/+ and IFN-R-/- mice were infected with LCMV-T1b, and primary virus-specific CD8 T cell responses were analyzed by intracellular cytokine staining on day 8 PI. As shown in Fig. 8, A and B, in the LCMV-T1b-infected +/+ mice, the immunodominance hierarchy of the LCMV-specific CD8 T cell response was GP33 > NP396 > GP276 GP118 NP205. Data in Fig. 8, A and B, show that the immunodominance hierarchy of the primary CD8 T cell response in LCMV-T1b-infected IFN-R-/- mice was comparable with +/+ mice. On day 8 PI, the LCMV-T1b titers in liver, lung, and kidney of +/+ and IFN-R-/- mice were comparable (data not shown). Attempts were made to examine the role of IFN-/IFN-R interactions in regulating the epitope hierarchy of the memory CD8 T cell pool after a chronic LCMV infection. However, LCMV-T1b-infected IFN-R-/- mice succumbed to the infection and failed to survive beyond 21 days PI. The death of LCMV-T1b-infected IFN-R-/- mice was likely due to CD8 T cell-mediated immunopathology (34).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
/IFN-R interactions in regulating the immunodominance hierarchy of the virus-specific CD8 T cell response in an acute or a chronic LCMV infection. As mentioned before, it is well documented that the epitope hierarchy of the primary CD8 T cell response is conserved during the phase of memory in a setting of acute viral infection (12, 13). In this study, we confirmed these findings and extended our knowledge about regulation of immunodominance hierarchy during chronic viral infection. Our studies showed that, unlike an acute LCMV infection, the immunodominance hierarchy of the primary response is not maintained in the memory CD8 T cell pool in mice that have resolved a chronic infection. After attaining a peak on day 8 PI, CD8 T cells undergo a contraction phase when large numbers of LCMV-specific CD8 T cells are eliminated, presumably by apoptosis (13). Our studies show that during a chronic LCMV infection, the contraction of GP276-specific CD8 T cells seems to be substantially attenuated, as compared with CD8 T cells specific to other LCMV CTL epitopes. Therefore, the alteration in the immunodominance hierarchy during a chronic LCMV infection is likely to result from differential susceptibility of CD8 T cells to deletion mechanisms based on epitope specificity. It was recently reported that the contraction phase of the CD8 T cell response might be programmed early after infection (35). Our studies suggest that programmed contraction of the CD8 T cell response during a chronic LCMV infection might be epitope dependent. Why is immunodominance hierarchy changing during the course of a chronic LCMV infection and not after an acute infection? Regulation of immunodominance in CD8 T cell responses is complex and might be affected by multiple factors, including the affinity of the antigenic peptide to the MHC, efficiency of processing of the antigenic epitope, temporal sequence of protein expression in infected cells, relative abundance of protein Ag in the APC, nature of APC, the presence of costimulatory interactions between T cell and APC, T cell repertoire, and clonal competition between T cells to interact with the APC (11). In the context of the present study, the factors that could be affecting the immunodominance are the antigenic load and/or the duration of infection. During an acute LCMV infection, it is possible that CD8 T cells specific to the dominant epitopes respond rapidly and reduce the antigenic load, thereby limiting the activation and expansion of subdominant epitope-recognizing CD8 T cells. However, during a chronic infection, contraction of CD8 T cells specific to the dominant epitopes might provide opportunities for activation and expansion of subdominant epitope-specific CD8 T cell clones. The alteration in epitope hierarchy seen in the memory phase after a chronic infection and not acute infection might be due to differences in cell tropism between LCMV-Arm and LCMV-T1b (25). However, increasing the dose of LCMV-Arm resulted in a significant change in the CD8 T cell epitope hierarchy of the memory T cell pool. Taken together, our studies show that the viral dose and/or duration of infection are critical parameters that regulate immunodominance hierarchy of the memory CD8 T cell pool during an acute LCMV infection. While this manuscript was in preparation, it was reported that persistently infected perforin-deficient mice (perf-/-) exhibit alteration in epitope hierarchy during the course of a persistent infection (36). Although this report is consistent with our data, the mechanisms underlying the alteration in immunodominance hierarchy in persistently infected perf-/- mice could be different. Our results on changing immunodominance during a chronic LCMV infection are largely in agreement with another recent report (37). The kinetics of Ag-specific CD8 T cell responses have also been studied in mice persistently infected with murine cytomegalovirus (38). Unlike in our studies, murine cytomegalovirus-infected mice do not show altered immunodominance but tend to accumulate virus-specific CD8 T cells over time.
Several studies have documented functional impairment of CD8 T cells during persistent viral infections of mice and humans (8, 16, 26, 36, 37). In these studies, the dysfunctional status of the CD8 T cells was associated with conditions of high viral load in the infected host. Several LCMV strains can cause a chronic infection in immunocompetent mice, but the duration of infection varies from one strain to another (21, 22, 23, 25). Unlike LCMV-T1b, which is resolved in 30 days, infection of mice with clone 13 strain of LCMV (LCMV-clone 13) results in a protracted infection lasting up to 6 mo (21). Both LCMV-T1b and LCMV-clone 13 were isolated from the lymphoid tissues of LCMV carrier mice infected at birth with LCMV-Arm (22, 23, 25), and both exhibit >99% genetic identity (23). Using LCMV-clone 13, it was recently reported that high viral load in the tissues is associated with the failure of a large proportion of virus-specific CD8 T cells to produce detectable levels of IFN-, TNF-, and IL-2 (37). This state of impairment in cytokine producing ability is termed as "full functional exhaustion." In LCMV-clone 13-infected mice, in some instances, this functional exhaustion can be incomplete (partial exhaustion), wherein CD8 T cells are able to produce low levels of IFN- but are unable to produce TNF- and IL-2 (37). In the present study, the functional impairment of CD8 T cells seen in LCMV-T1b-infected mice is distinct from the partially exhausted phenotype seen in LCMV-clone 13-infected mice. In LCMV-T1b-infected mice, CD8 T cells were able to produce both IFN- and TNF-, albeit at lower levels, as compared with LCMV-Arm-infected mice. Therefore, the functional status of CD8 T cells in LCMV-T1b-infected mice might reflect a "milder" form of partial exhaustion. It is worth emphasizing that, unlike the LCMV-clone 13 study, which was done in animals with high viral load (37), our experiments were performed several months after viral clearance. Hence, it is possible that CD8 T cells with a similar functional phenotype (milder exhaustion) can be detected in LCMV-clone 13-infected mice several months after viral clearance. Nonetheless, our findings show that chronic antigenic stimulation could lead to long-term functional defects in memory CD8 T cells. Further studies to investigate the processes of TCR signaling and cytokine gene transcription and translation in CD8 T cells from LCMV-T1b-infected mice might shed some light on the underlying defect in cytokine production.
We also examined whether memory CD8 T cells generated after the clearance of a chronic LCMV infection were phenotypically different from memory CD8 T cells in acutely infected mice. In our studies, memory CD8 T cells generated after an acute or chronic LCMV infection were uniformly CD44high. Occasionally, NP396-specific memory CD8 T cells show slightly reduced expression of CD44 in LCMV-T1b-infected mice, as compared with LCMV-Arm-infected mice. In contrast with our studies, virus-specific CD8 T cells in LCMV-clone 13-infected mice consistently expressed low levels of CD44, as compared with LCMV-Arm-infected mice (37). The underlying mechanisms and biological significance of altered CD44 expression in chronic LCMV infections needs further investigation. Interestingly, in the present study, there were notable differences in the fraction of CD62L expressing LCMV-specific memory CD8 T cells between acute and chronic infections. Although a large fraction of memory CD8 T cells in acutely infected mice were CD62Lhigh, the majority of memory CD8 T cells were CD62Llow after chronic infection. At the peak of the T cell response (day 8 PI) in both acute and chronic LCMV infection, LCMV-specific CD8 T cells are CD62Llow (data not shown). After viral clearance, these CD62Llow cells gradually revert to a CD62Lhigh phenotype (39). The rate of reversal of CD8 T cells from a CD62Llow to a CD62Lhigh phenotype seems to be determined by the duration and magnitude of antigenic stimulation (39). The persistence of CD62Llow phenotype of memory CD8 T cells in mice that have recovered from a chronic infection is reminiscent of prolonged antigenic stimulation in LCMV-T1b-infected mice, as compared with acutely infected mice. In acutely infected mice, there was an excellent correlation between CCR7 and CD62L expression on memory CD8 T cells. However, in mice that have recovered from a chronic infection, there was no correlation between CCR7 and CD62L expression in LCMV-specific memory CD8 T cells.
Similar to LCMV infection, the CD8 T cell response to intracellular bacteria LM is also directed against dominant andsubdominant epitopes (32). In LM infection of mice, the immunodominance hierarchy of the primary CD8 T cell response seems to be regulated by IFN- (32). Under conditions of IFN- deficiency, in LM-infected mice, the CD8 T cells recognizing the subdominant epitope exhibit increased expansion reaching levels comparable to that of the dominant epitope and subsequent loss of epitope hierarchy (32). These data suggest that IFN- could suppress the activation and expansion of subdominant epitope-specific CD8 T cells, at least in the LM infection of mice. In striking contrast to this report, our studies showed little to no alteration in the immunodominance hierarchy of the primary CD8 T cell response to LCMV in IFN-R-/- mice, as compared with +/+ mice. The differences in pathogenesis (cell tropism, antigenic load, temporal pattern of Ag expression in APCs, kinetics of CD8 T cell activation, the role of factors of innate immunity, etc.) between LM and LCMV infection might dictate the sensitivity of the immunodominance hierarchy of the primary CD8 T cell response to regulation by IFN-. In LCMV-Arm-infected +/+ mice, the epitope hierarchy of the primary CD8 T cell response and the pool of memory CD8 T cells were remarkably similar. In striking contrast, in IFN-R-/- mice, the epitope hierarchy of the pool of LCMV-specific memory CD8 T cells was substantially altered as compared with the primary response. IFN- or IFNR deficiency causes a slight delay in clearing an acute LCMV infection, as compared with +/+ mice (32, 40). Therefore, the observed alteration of immunodominance hierarchy among LCMV-specific memory CD8 T cells in IFN-R-/- mice could be a consequence of loss of IFN-R signaling and/or longer duration of antigenic stimulation, which needs further investigation.
In summary, we have demonstrated that increasing the viral dose or duration of infection can have significant effects on the CD8 T cell responses in vivo with special reference to: 1) regulation of immunodominance hierarchy during the phase of T cell memory, 2) functional competence of memory T cells, and 3) alterations in the expression of cell surface molecules on memory CD8 T cells. These findings might have significant implications in vaccine design and targeting of epitopes for immunotherapy during chronic viral infections
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 K.T. and J.S. contributed equally to this work.
3 Address correspondence and reprint requests to Dr. M. Suresh, Department of Pathobiological Sciences, University of Wisconsin, 2015 Linden Drive, Madison, WI 53706. E-mail address: sureshm{at}svm.vetmed.wisc.edu
4 Abbreviations used in this paper: LCMV, lymphocytic choriomeningitis virus; CD62L, L-selectin; MHC-I, MHC class I; PI, postinfection; LM, Listeria monocytogenes.
Received for publication April 7, 2003. Accepted for publication November 20, 2003.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
receptor. Science 259:1742.. Science 290:1354. interferon. J. Virol. 76:4251./ and interferons in persistence of lymphocytic choriomeningitis virus by clonal exhaustion of cytotoxic T cells. J. Virol. 75:8407. interferon receptor knockout mice. J. Virol. 72:7815.This article has been cited by other articles:
|
|
 |
|
  L. M. Bixby and R. L. Tarleton Stable CD8+ T Cell Memory during Persistent Trypanosoma cruzi Infection J. Immunol., August 15, 2008; 181(4): 2644 - 2650. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. N. Radcliffe, J. S. Roddick, F. K. Stevenson, and S. M. Thirdborough Prolonged Antigen Expression following DNA Vaccination Impairs Effector CD8+ T Cell Function and Memory Development J. Immunol., December 15, 2007; 179(12): 8313 - 8321. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Tewari, Y. Nakayama, and M. Suresh Role of Direct Effects of IFN-{gamma} on T Cells in the Regulation of CD8 T Cell Homeostasis J. Immunol., August 15, 2007; 179(4): 2115 - 2125. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. K. Whitmire, B. Eam, N. Benning, and J. L. Whitton Direct Interferon-{gamma} Signaling Dramatically Enhances CD4+ and CD8+ T Cell Memory J. Immunol., July 15, 2007; 179(2): 1190 - 1197. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Zhang, A. L. Cote, V. C. de Vries, E. J. Usherwood, and M. J. Turk Induction of Postsurgical Tumor Immunity and T-Cell Memory by a Poorly Immunogenic Tumor Cancer Res., July 1, 2007; 67(13): 6468 - 6476. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. F. Kotturi, B. Peters, F. Buendia-Laysa Jr., J. Sidney, C. Oseroff, J. Botten, H. Grey, M. J. Buchmeier, and A. Sette The CD8+ T-Cell Response to Lymphocytic Choriomeningitis Virus Involves the L Antigen: Uncovering New Tricks for an Old Virus J. Virol., May 15, 2007; 81(10): 4928 - 4940. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. H. Hogan, D. O. Co, J. Karman, E. Heninger, M. Suresh, and M. Sandor Virally Activated CD8 T Cells Home to Mycobacterium bovis BCG-Induced Granulomas but Enhance Antimycobacterial Protection Only in Immunodeficient Mice Infect. Immun., March 1, 2007; 75(3): 1154 - 1166. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. J. Obar, S. Fuse, E. K. Leung, S. C. Bellfy, and E. J. Usherwood Gammaherpesvirus persistence alters key CD8 T-cell memory characteristics and enhances antiviral protection. J. Virol., September 1, 2006; 80(17): 8303 - 8315. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Fuse, J. J. Obar, S. Bellfy, E. K. Leung, W. Zhang, and E. J. Usherwood CD80 and CD86 Control Antiviral CD8+ T-Cell Function and Immune Surveillance of Murine Gammaherpesvirus 68. J. Virol., September 1, 2006; 80(18): 9159 - 9170. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. C. Kemball, E. D. H. Lee, E. Szomolanyi-Tsuda, T. C. Pearson, C. P. Larsen, and A. E. Lukacher Costimulation Requirements for Antiviral CD8+ T Cells Differ for Acute and Persistent Phases of Polyoma Virus Infection J. Immunol., February 1, 2006; 176(3): 1814 - 1824. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. C. Kemball, E. D. H. Lee, V. Vezys, T. C. Pearson, C. P. Larsen, and A. E. Lukacher Late Priming and Variability of Epitope-Specific CD8+ T Cell Responses during a Persistent Virus Infection J. Immunol., June 15, 2005; 174(12): 7950 - 7960. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Suresh, A. Singh, and C. Fischer Role of Tumor Necrosis Factor Receptors in Regulating CD8 T-Cell Responses during Acute Lymphocytic Choriomeningitis Virus Infection J. Virol., January 1, 2005; 79(1): 202 - 213. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
