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The Journal of Immunology, 2006, 176: 3062-3069.
Copyright © 2006 by The American Association of Immunologists

Vaccine-Induced Memory CD8+ T Cells Cannot Prevent Central Nervous System Virus Reactivation1

Chandran Ramakrishna1,*, Roscoe A. Atkinson{dagger}, Stephen A. Stohlman2,*,{dagger},{ddagger} and Cornelia C. Bergmann2,3,*,{dagger}

* Department of Neurology, {dagger} Department of Pathology, and {ddagger} Department of Molecular Microbiology and Immunology, University of Southern California, Los Angeles, CA 90033


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Noncytopathic viruses use multiple strategies to evade immune detection, challenging a role for vaccine induced CTL in preventing microbial persistence. Recrudescence of neurotropic coronavirus due to loss of T cell-mediated immune control provided an experimental model to test T cell vaccination efficacy in the absence of Ab. Challenge virus was rapidly controlled in vaccinated Ab-deficient mice coincident with accelerated recruitment of memory CD8+ T cells and enhanced effector function compared with primary CD8+ T cell responses. In contrast to primary effectors, reactivated memory cells persisted in the CNS at higher frequencies and retained ex vivo cytolytic activity. Nevertheless, despite earlier and prolonged T cell-mediated control in the CNS of vaccinated mice, virus ultimately reactivated. Apparent loss of memory CD8+ effector function in vivo was supported by a prominent decline in MHC expression on CNS resident target cells, presumably reflecting diminished IFN-{gamma}. Severely reduced MHC expression on glial cells at the time of recrudescence suggested that memory T cells, although fully armed to exert antiviral activity upon Ag recognition in vitro, are not responsive in an environment presenting few if any target MHC molecules. Paradoxically, effective clearance of viral Ag thus affords persisting virus a window of opportunity to escape from immune surveillance. These studies demonstrate that vaccine-induced T cell memory alone is unable to control persisting virus in a tissue with strict IFN-dependent MHC regulation, as evident in immune privileged sites.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Effective antimicrobial vaccines induce immune effector functions that provide sustained protective memory. However, the ability of viruses to establish persistent infections and reactivate is a major challenge for successful vaccine candidates (1). Sustained CD8+ T cell surveillance and control of persisting viruses during the natural course of infections raises hope that vaccines inducing T cell responses may be feasible to combat persisting viruses (2, 3). Viruses that persist, such as HIV, herpes viruses, papilloma virus, and hepatitis B and hepatitis C viruses use diverse immune evasion strategies (1, 3, 4). These include T cell and Ab escape variants (5, 6), viral mediated inhibition of Ag processing (1), T cell exhaustion and physical deletion (7, 8, 9, 10). Infection of sites not readily accessible to the immune system, i.e., the eye, CNS, and testis (11, 12), constitutes an additional pathogen survival strategy. Factors contributing to the CNS as a favored site of viral persistence include stringent requirements for MHC presentation, absence of lymphoid tissue and a well-defined lymphatic drainage, the blood brain barrier that restricts lymphocyte trafficking, and secretion of anti-inflammatory substances (11). CNS virus reactivation resulting from ineffective immunological surveillance, although relatively uncommon, is potentially devastating (11, 13, 14, 15, 16, 17). In addition, preferential use of noncytolytic immune mechanisms to eliminate pathogens, thereby avoiding neurological damage (11), can result in nonsterilizing immunity and facilitate viral persistence. Terminally differentiated long-lived CNS cells express few MHC molecules and appear refractory to viral or CTL induced apoptosis, therefore constituting efficient long-term reservoirs (11, 18). These complex regulatory interactions suggest that even optimal CD8+ T cell responses may potentially fail to prevent and control viral persistence in immune privileged organs such as the CNS.

Control of a noncytopathic neurotropic coronavirus infection was examined to provide insights into the efficacy of T cell-based vaccines in combating the establishment of persistent infection restricted to the CNS (19, 20). CNS replication of the mouse hepatitis virus JHM strain (JHMV)4 is controlled by CD8+ T cells via perforin and IFN-{gamma}-dependent mechanisms (19, 20, 21). A minimal role for Ab during acute infection was confirmed by efficient virus control in the CNS of B cell-deficient (JHD) mice (22). However, despite initial virus control and retention of CD8+ T cells in the CNS, virus recrudesces in the absence of humoral immunity (22). Apparent loss of CD8+ T cell-mediated control of recrudescing virus is associated with loss of cytolytic function (22). Although T cell exhaustion (4, 7, 8, 9, 10), impaired Ag presentation (1), the immunoregulatory CNS environment (11), or any combination of these mechanisms may further contribute to virus recrudescence, CTL escape mutants evident in other infections (5, 6) do not appear to play a role in adult JHMV-infected mice (23, 24). In contrast to loss of cytolytic activity by CD8+ T cells retained in the CNS following acute infection, reactivated memory CD8+ T cells recruited into the CNS eliminate virus more rapidly and retain effector function (25, 26). These data provided the basis to test whether vaccine-induced memory T cells suffice to limit viral replication, thereby preventing viral persistence and subsequent reactivation in the absence of humoral immunity.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice, viruses, and immunizations

Primary (1') CD8+ T cells were induced by infecting age-matched naive B cell-deficient JHD mice (H-2d) with 500 PFU of neurotropic JHMV intracerebrally as described (22). Secondary (2') CD8+ T cell responses were elicited by intracerebral infection of JHD mice immunized i.p. with 5 x 106 PFU of JHMV 4 wk earlier. The absence of Ab in these mice allowed the evaluation of memory CD8+ T cell responses without compromising the ability to establish an efficient CNS infection. All procedures were performed in compliance with Keck School of Medicine (University of Southern California, Los Angeles, CA) Institutional Animal Care and Use Committee approved protocols. In immunized mice 8–10% of splenic CD8+ T cells were specific for the dominant nucleocapsid protein epitope, pN, at the time of challenge, and no virus specific CD8+ T cells were detected in the CNS before challenge. Infectious JHMV in clarified brain homogenates was determined by plaque assay as described (22).

Clinical disease

Clinical disease was scored as previously described (27). Briefly, mice were graded as 0 = healthy; 1 = ruffled hair and hunchbacked appearance; 2 = reduced mobility and inability to upright; 3 = paralysis and wasting; and 4 = death. Data represent average of at least four mice per time point and are representative of three or more experiments for each group of mice.

Isolation of mononuclear cells

CNS-derived mononuclear cells (CMC) were isolated as previously described (22). Briefly, brains were removed from mice perfused with PBS, homogenized in RPMI 1640 containing 25 mM HEPES (pH 7.2), using Tenbroek homogenizers and adjusted to 30% Percoll (Amersham Biosciences). To isolate oligodendroglia, brains and spinal cords were obtained from perfused mice and digested with 0.25% trypsin as previously described (28). CMC were concentrated by centrifugation at 800 x g for 25 min at 4°C onto a 70% Percoll cushion. Cells were recovered from the 30%/70% interface and washed twice before analysis.

CTL assays

CMC were evaluated for ex vivo cytolytic activity as previously described using Ld restricted immunodominant pN peptide (29) coated J774.1 (H-2d) target cells labeled with Na51CrO4 (22, 29). Spontaneous release was <20% of total release in all experiments. Specific lysis per tetramer+CD8+ T cell was calculated by adjusting E:T ratios to tetramer+CD8+ T cells.

Flow cytometry

Single-cell suspensions obtained from CNS or spleen were blocked with a mixture (10%) of mouse, rat, and human sera (Atlanta Biologicals) and rat anti-mouse CD16/CD32 (2.4G2; BD Pharmingen) before incubation with a variety of Abs to determine surface expression. PE, FITC, PerCP, and allophycocyanin labeled mAb specific for CD45 (30-F11), CD4 (RM4-5), CD8 (53-6.7), CD44 (IM7), CD62 ligand (MEL-14), CD43 (S7), Ly6G (1A8), MHC class I (39-10-8), and MHC class II (2G9) were all obtained from BD Pharmingen. Anti-F4/80 PE was obtained from Serotec. JHMV-specific CD8+ T cells were identified by Ld-pN MHC class I tetramers as previously described (29). The function of pN-specific CD8+ T cells was determined by intracellular cytokine staining for IFN-{gamma} and TNF-{alpha} using conjugated Ab obtained from BD Pharmingen as previously described (25). All cells were analyzed with a FACSCalibur flow cytometer (BD Biosciences) using CellQuest Pro software. CD45 (30-F11; BD Pharmingen) expression was used to distinguish bone marrow-derived infiltrating cells (CD45high), CNS resident microglia (CD45low), and other CNS resident cells (CD45–). Oligodendrocytes in the CD45– population were identified using biotinylated O4 mAb followed by avidin/allophycocyanin (BD Pharmingen) as described (28). The gating strategy distinguishing infiltrating bone marrow-derived CD45high leukocytes from CD45low microglia and CD45– O4+ oligodendrocytes is shown in Fig. 7A.


Figure 7
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  		FIGURE 7. Diminished MHC class I expression on CNS resident cells of infected 2' mice. A, Representative density plots of CD45 and class I expression in the acutely infected CNS (top plot). CD45 expression distinguishes infiltrating CD45high leukocytes (region R2) from CD45low O4– microglia (R3) and CD45– O4+ oligodendroglia (R4). Density plot (bottom) depicts O4 and class I staining from CD45– cells derived from the R4 gate in the top plot. O4+ oligodendroglia are contained within the highlighted R5 region. B, Representative density plots of class I expression on CD45high CNS infiltrates and CD45low microglia (see A) from infected 1' and 2' mice at the indicated time points p.i. C, Representative density plots of CNS resident CD45– O4+ oligodendroglia (see R5 in A) expressing class I from infected 1' (days 8 and 45 p.i.) and 2' (days 8 and 60 p.i.) mice. Data obtained at day 60 p.i. for 2' mice was derived from sick mice with clinical disease scores of 2.0–2.5. Oligodendroglia from naive mice are class I–. Values represent percentages of class I+ cells, and mean fluorescence intensity is shown in parenthesis.

 
Histology

Brains, bisected in the midcoronal plane, and spinal cords were examined for inflammation and distribution of viral Ag. Tissues were fixed for 3 h in Clark’s solution (75% ethanol, 25% glacial acetic acid) before embedding. Sections were stained with H&E to determine inflammation. Distribution of viral Ag was determined by immunoperoxidase staining (Vectastain ABC kit; Vector Laboratories) using the anti-JHMV mAb J.3.3 specific for the C terminus of the viral nucleocapsid protein as the primary Ab (27) and horse anti-mouse as secondary Ab (Vector Laboratories). Sections were scored for inflammation and viral Ag in a blinded fashion. Representative fields were identified based on average scores of all sections in each experimental group.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Rapid CNS inflammation in immunized mice

Memory CD8+ T cells were induced via a self limiting peripheral JHMV infection of B cell-deficient JHD mice. Vaccinated mice were subsequently challenged with JHMV (2' mice) and inflammatory responses as well as control of virus replication compared with infected naive mice (1' mice). Infection induced an accelerated and increased inflammatory CNS response in vaccinated mice, consistent with the rapid mobilization of memory T cells in response to infection (25, 26, 30, 31). Inflammatory cells, characterized by a CD45high phenotype, were increased 5-fold within the CNS of vaccinated mice compared with naive mice at day 5 postinfection (p.i.) (Fig. 1A). Despite this rapid onset, CNS inflammation in 1' and 2' responders was similar by day 8 p.i. and remained at equivalent levels thereafter (Fig. 1A). The composition of CD45high infiltrates was analyzed throughout infection to determine cell types preferentially recruited. Macrophages constituted the major component (~40%) in the CNS infiltrates of both groups at day 5 p.i., but their proportion declined below <20% thereafter (data not shown). The relative frequencies of NK cells and Ly-6G+ neutrophils were reduced in 2' CNS infiltrates compared with 1' infiltrates at day 5 p.i. (data not shown), but their total numbers were similar given the 5-fold increase in CD45high infiltrating cells (Fig. 1A).


Figure 1
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  		FIGURE 1. Rapid CNS mononuclear infiltration in infected 2' responders. Immune JHD mice harboring virus-specific T cells (2') and age-matched naive JHD mice (1') were challenged with JHMV intracerebrally. Single-cell suspensions were prepared from the CNS of infected 2' and 1' mice (n = 4–5/time point). A, Data represent the total number of CD45high infiltrates recovered per brain from 2' ({blacksquare}) and 1' ({square}) mice. B, CD8+ T cell percentages within 2' ({blacksquare}) and 1' ({square}) CD45high populations. Data were calculated by setting CD45high infiltrating cells to 100%. Values atop columns denote relative percentages of Ld/pN tetramer+ cells within CD8+ T cell populations. C, Total number of tetramer+CD8+ T cells per brain of 2' ({blacksquare}) or 1' ({square}) mice. Data represent an average of two to three experiments. nd, Not done due to high mortality (>95%) of 1' mice by day 60 p.i. Error bars indicate SEM.

 
CD8+ T cells within the CNS infiltrates of 2' mice constituted a prominent population (35%), compared with only ~10% in 1' mice at day 5 p.i. (Fig. 1B), and continued to comprise a dominant and stable population (~60–70%) throughout infection. More importantly, ~45% of CD8+ T cells within the CNS of 2' mice were already virus-specific at day 5 p.i. and further increased to a stable fraction of ~70% by day 8 p.i. (Fig. 1B). By contrast, virus-specific CD8+ T cells in the CNS of 1' mice were a minor fraction at day 5 p.i., and only reached a maximum of ~40% between days 8 and 25 p.i. (Fig. 1B). These data are consistent with rapid recruitment of both pathogen-specific and irrelevant T cells into the infected CNS, but preferential retention of Ag-specific CD8+ T cells (32, 33, 34).

Comparison of total numbers of virus-specific CD8+ T cells in 1' and 2' responders dramatically emphasized differences in mobilization and CNS recruitment. Virus-specific CD8+ T cells in the CNS of 1' responders were at the limit of detection at day 5 p.i. and accumulated to a maximum of ~1.3 x 105 at day 8 p.i. (Fig. 1C). The latter numbers of virus-specific CD8+ T cells were already present in the CNS of 2' responders by day 5 p.i. and further increased 3-fold at day 8 p.i. (Fig. 1C). Increased CNS recruitment in vaccinated mice correlated with enhanced virus-specific CD8+ T cell retention (Fig. 1C). CD8+ T cells localized in the CNS underwent a contraction phase following both 1' and 2' responses (Fig. 1C), albeit only to a modest extent compared with peripheral infections (35, 36). Although CD4+ T cells comprise a major component of the early response during primary infection (22, 25), they constituted <10% of infiltrates in the CNS of 2' mice (data not shown). JHMV infection of B cell-deficient JHD mice is not associated with skewing of CD8+ and CD4+ T cell memory populations (22). Furthermore, immunization of JHD mice results in similar frequencies of virus-specific memory CD4+ T cells compared with wild-type mice (data not shown). The meager accumulation of CD4+ T cells in the 2' CNS may thus result from preferential expansion of 2' CD8+ T cells following challenge.

Memory T cells induce rapid viral clearance but do not prevent recrudescence

Rapid mobilization of memory CD8+ T cells predicted enhanced control of virus replication and spread as well as potential suppression of viral recrudescence (22, 25). Virus replication in 2' mice was indeed reduced at day 5 p.i. compared with naive mice. Infectious virus declined to below detection in the majority (>60%) of 2' mice by day 8 p.i. (Fig. 2A) and was no longer detectable at day 14 p.i. Immunohistochemical analysis confirmed fewer viral Ag+ cells in the CNS of 2' mice at day 8 p.i. compared with the CNS of 1' mice (22) and by day 14 p.i. no viral Ag could be detected in the CNS of 2' mice (data not shown). By contrast, virus still replicated extensively in the CNS of 1' mice at day 8 p.i. (Fig. 2A) and clearance was delayed coincident with delayed virus-specific CD8+ T cell recruitment (Fig. 1C). Recrudescing infectious virus in the CNS of 1' mice results in increasing encephalomyelitis (Fig. 2B) and mortality (Fig. 2C and Ref.22). The rapid reduction in infectious virus and increased retention of virus-specific CD8+ T cells within the CNS of 2' mice coincided with a prolonged period of viral control (Fig. 2A). Nevertheless, infectious virus began to reemerge in the CNS of ~40% of vaccinated mice at day 45 p.i., and by day 60 p.i. ~90% had lost immunological control (Fig. 2A). The delay in reactivation is consistent with the inability to detect residual viral Ag+ cells at day 14 p.i. in 2' mice and suggests fewer numbers of persistently infected cells initiate recrudescence compared with 1' mice. Sequence analysis of reemerging viral RNA revealed no evidence for selection of mutations within the immunodominant CD8+ T cell epitope, excluding CTL escape variants (data not shown).


Figure 2
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  		FIGURE 2. Memory T cells do not prevent viral recrudescence. JHMV replication in the CNS of 1' and 2' JHD mice assessed by plaque assay at indicated time points p.i. A, Data represent mean CNS titers of at least three experiments with three to four mice per time point. Data obtained at day 60 p.i. for 2' mice was derived from sick mice with clinical disease scores of 2.0–2.5. Data were not obtained for 1' mice at day 60 p.i. due to >95% mortality. Morbidity (B) and mortality (C) in infected 1' and 2' JHD responder mice are shown. Data represent average of at least three separate experiments with at least eight mice per time point. Error bars indicate SEM.

 
The delay in viral recrudescence was reflected in reduced clinical disease and mortality in 2' JHD mice (Fig. 2, B and C). Although both groups survived acute infection, 1' mice had increasing mortality rates concomitant with virus recrudescence following 14 days p.i.; <8% survived following 45 days p.i. consistent with previous studies (22). Survival of 2' mice was prolonged, with ~50% of mice still surviving at 60 days p.i. (Fig. 2C). Relapse in clinical disease and increased mortality coincided with virus recrudescence in ~50% of 2' mice by 45 days p.i. (Fig. 2). Mice with increased clinical disease at days 45 and 60 p.i. also had increased infectious virus in the CNS compared with those with milder clinical disease, supporting a correlation between viral load and mortality. Overall, viral titers and clinical scores remained lower in vaccinated mice during recrudescence before mortality. Thus, memory T cells are highly effective in controlling a secondary infection; however, they are ultimately unable to prevent CNS persistence and only delay viral reactivation.

Memory T cells cannot prevent virus reactivation in oligodendrocytes

Immunohistochemical analysis was performed on brains and spinal cords derived from both groups at 45 days p.i. to reveal Ag localization during virus recrudescence. Virus Ag was detected in all cell types infected during acute infection, i.e., astrocytes, microglia, and oligodendrocytes in brains (data not shown) and spinal cords of 1' mice (Fig. 3A), consistent with a previous report (22). By contrast, recrudescing virus localized only to oligodendroglia in both brains (data not shown) and spinal cords of 2' mice at day 45 p.i. (Fig. 3B). Preferential oligodendroglial tropism was still evident at day 60 p.i. in the CNS of 2' JHD mice during virus recrudescence, implying that oligodendroglia are preferentially infected or are more resistant to CD8+ T cell effector function than astrocytes and microglia.


Figure 3
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  		FIGURE 3. Memory T cells limit viral Ag to oligodendrocytes. JHMV Ag in the spinal cords of infected 1' (A) and 2' (B) responder mice (immunoperoxidase stain for JHMV Ag with hematoxylin counterstain; magnification is x200) at day 45 p.i. In 2' responders, viral Ag is limited to a small number of cells identified morphologically as oligodendrocytes (arrowheads). In 1' responders, JHMV Ag-positive cells were more numerous and detected in astrocytes (arrows) and oligodendrocytes (arrowheads). Scale bar, 100 µm. *, gray matter/white matter junction.

 
Reactivated memory cells within the CNS retain effector function following ex vivo Ag stimulation

CD8+ T cells within the CNS lose cytolytic function following clearance of infectious virus in 1' responders, independent of clinical disease, pathology, or virus recrudescence (20, 22, 29). By contrast, 2' CD8+ T cells recruited into the CNS retain cytolytic activity in the absence of infectious virus (22, 25). Ex vivo cytolysis by cells derived from the CNS of 2' and 1' mice was compared to determine whether cytolytic activity was also retained by 2' CD8+ T cells under conditions of increasing infectious virus. Cells from 2' mice exhibited increased cytolytic activity at day 8 p.i., not only at the population level but also on a single cell basis (Fig. 4). Importantly, unlike 1' CD8+ T cells, 2' CD8+ T cells continued to express cytolytic activity out to 60 days p.i. (Fig. 4). IFN-{gamma} is critical for both efficient Ag presentation by CNS resident cells and control of infectious virus in oligodendrocytes (21, 37, 38). Virus-specific IFN-{gamma} secreting CD8+ T cells were prominent by day 5 p.i. in the 2' CNS and increased dramatically by day 8 p.i. (Fig. 5). The relative number of IFN-{gamma}-secreting CD8+ T cells within the CNS of both 1' and 2' mice remained constant after day 14 p.i., suggesting the capacity to secrete IFN-{gamma} was not compromised during virus recrudescence (Fig. 5). Therefore, activated memory cells persisting within the CNS for 2 mo after apparent viral control retain both IFN-{gamma} secreting and cytolytic effector functions, but yet are unable to prevent viral reactivation.


Figure 4
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  		FIGURE 4. 2' CD8+ T cells retain cytolytic potential. Ex vivo cytolytic activity of CMC from JHMV-infected 2' and 1' mice (n = 5–7) analyzed using peptide-coated J774.1 (H-2d) target cells at the indicated days p.i. Numbers in parenthesis denote the ratio of tetramer+CD8+ T cells to target cells. Data represent one of two similar experiments. Far right, Specific cytolysis normalized to reflect lysis achieved by one tetramer+ cell per target.

 

Figure 5
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  		FIGURE 5. 2' CD8+ T cells remain functional during virus recrudescence. Data represent total numbers of IFN-{gamma}+CD8+ T cells per brain of JHMV infected 2' ({blacksquare}) and 1' ({square}) mice at indicated times p.i. Data calculated based on total cell yields and represent the average of three to five independent experiments. nd, Not done due to >96% mortality in 1' mice at day 60 p.i. Error bars indicate SEM.

 
Absence of in vivo T cell function correlates with inefficient Ag presentation

Expression of cytolysis and IFN-{gamma} production following Ag stimulation in vitro indicated that the inability of virus-specific CD8+ T cells to prevent recrudescence was not the result of anergy, but rather ineffective in vivo Ag recognition. Cytokine secretion by T cells is Ag-specific and dependent on TCR-MHC engagement (39). Although MHC class I is not expressed by resting CNS cells (38), microglia and oligodendroglia up-regulate MHC class I during JHMV-induced inflammation (21, 40) providing targets for T cell engagement. IFN-{gamma} is a key factor in enhancing class I surface expression and is essential in inducing class II expression on microglia during JHMV infection (21, 41). However, the longevity of MHC expression once virus is controlled is unknown. MHC expression persists on microglia (~50%) for at least 1 wk following clearance of infectious virus from wild-type mice (data not shown). Rare TCR-ligand interactions in vivo, as a result of rapid viral control by memory CD8 T cells, could severely limit IFN-{gamma} production (39, 42). IFN-{gamma} secretion in vivo was thus indirectly monitored via IFN-{gamma}-dependent MHC class II expression on microglia (21). MHC class II up-regulation was more prominent on microglia from 2' mice compared with 1' mice at day 5 p.i. (Fig. 6A). At day 8 p.i., although the frequency of class II+ microglia was similar in both 1' and 2' mice, the expression levels were higher in vaccinated mice (Fig. 6), consistent with patterns of virus-specific CD8+ T cell recruitment (Fig. 1C). Microglial class II expression was retained in 1' mice during virus recrudescence (days 25–45 p.i.) (Fig. 6), suggesting that sufficient virus Ag was presented in vivo to sustain IFN-{gamma} secretion during the period between virus control and recrudescence. However, class II expression on microglia from 2' mice gradually diminished (Fig. 6) following virus clearance and did not increase during virus recrudescence. These data imply that rapid clearance of viral Ag in 2' mice results in reduced TCR-MHC mediated IFN-{gamma} secretion and subsequent loss of class II expression, reminiscent of in vivo results in the lymphocytic choriomeningitis virus model (42). These results were supported by measuring IFN-{gamma} protein levels by ELISA in brain homogenates. Both groups of mice expressed high levels of IFN-{gamma} (~25 ng/brain) at day 7 p.i. (data not shown). However, although IFN-{gamma} could not be detected following day 10 p.i. in 2' CNS supernatants, low levels of IFN-{gamma} could be detected even at day 30 p.i. in brains of 1' mice, consistent with prolonged microglial MHC class II expression.


Figure 6
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  		FIGURE 6. Diminished IFN-{gamma} secretion by 2' CD8+ T cells in vivo but not in vitro during virus recrudescence. A, Percentages of class II expressing CD45low microglia in brains of 2' ({blacksquare}) and 1' ({square}) infected mice. B, Histograms gated on CD45low microglia (see Fig. 7A, R3 region). Microglia from naive mice (gray line histogram) or infected 1' (light dotted histogram) and 2' (dark solid histogram) mice during acute infection (day 8 p.i.) (bottom left) or virus (1', day 45; 2', day 60) (bottom right) recrudescence. M1 based on isotype control. Value represents percentage of class II+ microglia, and mean fluorescence intensity of class II expression is shown in parenthesis.

 
Microglia were also monitored for class I expression to examine how reduced viral Ag affected class I regulation during JHMV recrudescence because MHC class I expression is less dependent on IFN-{gamma} than MHC class II (21). Microglia and oligodendrocytes were distinguished by their CD45lowO4– and CD45–O4+ phenotypes, respectively (Fig. 7A). Microglia from both groups expressed high levels of MHC class I at day 8 p.i. (Fig. 7B). Similar to class II, class I expression was retained on microglia from 1' mice during recrudescence (Fig. 7B). By contrast, it declined on microglia from 2' mice following day 14 p.i. and was nearly completely extinguished during viral recrudescence at day 60 p.i. (Fig. 7B). Oligodendroglia, frequent reservoirs of persisting virus (18, 19, 43), also up-regulated class I during acute inflammation (Fig. 7C). Similar to microglia, oligodendroglia derived from 1' mice continued to express class I (Fig. 7C). By contrast, no class I was detected on oligodendroglia derived from 2' mice during recrudescence (Fig. 7C). Class I expression declined more rapidly in oligodendrocytes with only 50% expressing class I by day 21 p.i., when the vast majority of microglia still exhibited class I expression (data not shown). Class I Ag presentation in oligodendroglia may thus be more stringently regulated. These data suggest that the inability of CD8+ T cells to control recrudescence in 1' mice is due to impaired cytolytic function rather than inefficient MHC presentation. The inability to control CNS viral persistence in 2' mice, however, may be attributed to loss of MHC class I expression on CNS resident cells as a consequence of more effective clearance and fewer residually infected cells to sustain Ag-mediated IFN-{gamma} secretion compared with 1' JHD mice. As a paradoxical result, fully functional CD8+ T cells cannot exert effector function providing a window of opportunity for the virus to evade CD8+ T cell surveillance.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Vaccine-induced immunological memory attempts to recapitulate the effectors controlling acute infection to afford protection. However, mechanisms combating acute viral infections can differ, depending on route of entry, the target tissue, and cytopathicity. In general, humoral immunity is critical for control of cytopathic viruses and viruses that infect via the hematogenous route (44). By contrast, CD8+ T cells play a dominant role in controlling solid tissue infections and those induced by noncytopathic viruses including HIV, hepatitis B virus, hepatitis C virus, lymphocytic choriomeningitis virus, and JHMV (2, 3, 4, 19, 45, 46). The predominant lytic CD8+ T cell effector mechanism is perforin-dependent, but can also involve Fas-Fas ligand interactions (47). In addition, CD8+ T cells secrete a variety of effector molecules upon TCR engagement with MHC class I peptide complexes (39, 48, 49, 50). Due to their potential to induce damage, especially to cells with limited renewal capacity, CD8+ T cells are under strict regulatory control. The down-regulation of effector function as infection is controlled may facilitate viral persistence. In addition to reduced cytolytic potential, virus-infected cells escape CTL recognition by expressing altered epitopes, interfering with Ag processing or inhibiting class I expression (1). Secretion of inhibitory molecules, either induced by the virus itself or a physiological component of the target tissue, may also contribute to viral persistence.

Viruses that persist have evolved to occupy niches relatively inaccessible to immune effectors, considered to be immunologically privileged sites. Examples include papilloma virus and herpes virus infections of humans (15, 16, 18) and Theiler’s murine encephalomyelitis virus, lymphocytic choriomeningitis virus, and JHMV infections of mice (17, 19, 51). These viruses readily produce persistent infections in hosts with apparently competent immune effectors. Although the resting CNS is considered immunologically privileged, multiple innate and adaptive immune cells are recruited following infection (11). During acute coronavirus-induced encephalomyelitis, CD8+ T cells are the primary effectors of viral control (19, 20). As the host controls virus and Ag load decreases, cytolytic activity is lost (20, 22, 29), although the ability to secrete IFN-{gamma} is retained (11, 22). The loss of cytolytic activity contributes to the host’s inability to provide sterile immunity resulting in persistence characterized by viral Ag, viral RNA, but no detectable infectious virus (11, 19, 20). CD8+ T cells are retained in the persistently infected CNS expressing a quasi-activated surface phenotype (22, 29), but are unable to control virus reactivation (22). The inability to re-express cytolytic activity, even in the presence of increasing Ag (22), suggests a direct correlation between cytolytic capability and immune control of CNS viral persistence.

Data from two viral models indicated that memory cells, in stark contrast to 1' CD8+ T cells recruited into the CNS, retain cytolytic activity, even in the absence of viral persistence (25, 26). Therefore, the goal of this study was to determine whether enhanced effector functions by memory CD8+ T cells suffice to prevent virus persistence using a viral recrudescence model in B cell-deficient mice (22). Although analysis of CD8+ T cell-mediated elimination of allografts suggested impaired recruitment into immunologically privileged sites (52), the present data reveal amplified recruitment and expression of antiviral effector functions by memory T cells compared with newly activated T cells. Analysis of the CNS as a target organ of viral infection is thus consistent with the rapid activation, migration, and elimination of virus by memory cells in nonprivileged tissue sites (3, 30, 31).

Reactivated memory CD8+ T cells delayed, but could not prevent viral recrudescence, despite retention in the CNS at high numbers compared with those retained following primary responses. Delayed recrudescence correlated with the initial increase in antiviral effector function, which was evident by reduced viral replication as well as low frequencies of viral Ag+ cells during the acute phase. The vastly increased proportion of effector CD8+ T cells relative to persistently infected cells prolonged virus control in vaccinated mice. Reactivated memory CD8+ T cells within the CNS retained both cytolytic activity and IFN-{gamma} secretion ex vivo. Thus, virus recrudescence was not the result of Ag-induced anergy, which was demonstrated in other models of viral persistence (1, 7, 8, 9, 10). Sequence analysis of reactivated virus also revealed no evidence for CTL escape mutants (23, 24) associated with JHMV reactivation in a model of neonatal CNS infection (53). Furthermore, similar activation and function of virus-specific CD8+ and CD4+ T cells in infected JHD compared with B cell-sufficient, but Ab-deficient mice (22), ruled out a role for B cells in expansion of 2' CD8+ T cells. Although no defect in virus-specific CD4+ T cell memory was evident in JHMV vaccinated mice, impaired function within the CNS following challenge cannot be ruled out.

These data rather suggest that highly stringent MHC regulation by CNS resident cells underlies the escape from CD8+ T cell effector function. Thus, although reactivated memory CD8+ T cells in the CNS persisted in an armed state, the rapid reduction in viral load may have resulted in too few residual cognate recognition structures to sustain IFN-{gamma} production and consequently MHC expression. This concept was supported by the ability to detect IFN-{gamma} by ELISA in brain homogenates of 1' mice, but not 2' mice throughout recrudescence, as well as the loss of IFN-{gamma}-dependent class II expression (21) on microglia in 2' but not 1' responders. This apparent requirement for Ag recognition is consistent with the limited in vivo expression of IFN-{gamma} by memory CD8+ T cells (42). Although, the extent to which IFN-{gamma} regulates class I Ag presentation in glial cell subsets in vivo is unknown, our data indicate that sustained class I expression on CNS resident targets is also highly dependent on IFN-{gamma}. This is substantiated by increased and prolonged microglial class I expression in wild-type compared with IFN-{gamma}-deficient mice (21). Furthermore, although class I is up-regulated poorly on microglia in the absence of IFN-{gamma} (21), preliminary data analyzing infection of IFN-{gamma}-deficient mice suggest the absence of class I up-regulation on oligodendrocytes (C. C. Bergmann, unpublished observation). The half-life of surface class I molecules on glial cells is also unexplored. MHC is only slowly down-regulated even after clearance of infectious virus in wild-type mice. Thus the time interval between "clearance" and recrudescence in 1' JHD mice is insufficient to achieve the extent of class I down-regulation seen in 2' mice. This preempts a direct comparison of in vivo T cell function during the early stages of recrudescence. Furthermore, earlier down-regulation and reduced class I expression on oligodendrocytes, compared with microglia, before recrudescence in 2' mice may reflect reduced IFN-{gamma} mediated signaling (28). These findings highlight the concept that release of CD8+ T cell effector molecules such as perforin and IFN-{gamma} is not only strictly dependent upon recognition of cognate Ag-MHC structures in vitro, but also in situ (39, 42). Furthermore, comparison of 1' and 2' mice undergoing reactivation with equivalent CNS infectious virus loads, i.e., 1' day 25 p.i. and 2' day 60 p.i., suggests that the inability of 1' mice to control infectious virus results in virus replication in both oligodendroglia and other CNS cell types. By contrast, in the CNS of 2' mice delayed yet increasing virus recrudescence appears confined to oligodendroglia. The observation that class I remains undetectable on these glial cells suggests CD8+ T cells are unable to exert antiviral control due to the absence of recognition structures. However, it cannot be excluded that differential expression of costimulatory molecules or inhibitory ligands by oligodendrocytes compared with other glia may contribute to the apparent ignorance of virus-specific CD8+ T cells in vivo. Whether or not the low expression of class I on microglia results in the clearance of infectious virus from this cell type, limiting the overall levels of infectious virus within the CNS is unclear. In summary, vaccine-induced memory CD8+ T cells provide a rapid and apparently sustained antiviral response. However, even though infection appeared to be eliminated, virus reactivated within the CNS during a window in which MHC expression was not sustained and only few cells harbored persisting virus. Despite random distribution of CD8+ T cells, rare recognition of adjacent target cells would restrict IFN-{gamma} signaling to the local environment. Overall, the data suggest that an effective multivalent vaccine approach, especially directed toward noncytopathic viruses, may require induction of polyvalent memory responses combining both cellular and humoral components.


   Acknowledgments
 
We thank Emanuel Dimacali for assistance with flow cytometry and Eva Boruka for maintenance of the JHD colony.


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


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

1 This work was supported by National Institutes of Health Grants NS18146, NS40667, and AI47249. Back

2 Current address: Department of Neurosciences, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH 44195. Back

3 Address correspondence and reprint requests to Dr. Cornelia C. Bergmann, Department of Neurosciences, Lerner Research Institute, Cleveland Clinic Foundation, 9500 Euclid Avenue, NC30, Cleveland, OH 44195. E-mail address: bergmac{at}ccf.org Back

4 Abbreviations used in this paper: JHMV, JHM strain of mouse hepatitis virus; 1', primary; 2', secondary; CMC, CNS-derived mononuclear cell; p.i., postinfection. Back

Received for publication September 14, 2005. Accepted for publication December 13, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Tortorella, D., B. E. Gewurz, M. H. Furman, D. J. Schust, H. L. Ploegh. 2000. Viral subversion of the immune system. Annu. Rev. Immunol. 18: 861-926. [Medline]
  2. Robinson, H. L., R. R. Amara. 2005. T cell vaccines for microbial infections. Nat. Med. 11: (4 Suppl.):S25-S32. [Medline]
  3. Shoukry, N. H., A. G. Cawthon, C. M. Walker. 2004. Cell-mediated immunity and the outcome of hepatitis C virus infection. Annu. Rev. Microbiol. 58: 391-424. [Medline]
  4. Chisari, F. V., C. Ferrari. 1995. Hepatitis B virus immunopathogenesis. Annu. Rev. Immunol. 13: 29-60. [Medline]
  5. Borrow, P., G. M. Shaw. 1998. Cytotoxic T-lymphocyte escape viral variants: how important are they in viral evasion of immune clearance in vivo?. Immunol. Rev. 164: 37-51. [Medline]
  6. Ciurea, A., L. Hunziker, R. M. Zinkernagel, H. Hengartner. 2001. Viral escape from the neutralizing antibody response: the lymphocytic choriomeningitis virus model. Immunogenetics 53: 185-189. [Medline]
  7. Reignat, S., G. J. Webster, D. Brown, G. S. Ogg, A. King, S. L. Seneviratne, G. Dusheiko, R. Williams, M. K. Maini, A. Bertoletti. 2002. Escaping high viral load exhaustion: CD8 cells with altered tetramer binding in chronic hepatitis B virus infection. J. Exp. Med. 195: 1089-1101. [Abstract/Free Full Text]
  8. Gruener, N. H., F. Lechner, M. C. Jung, H. Diepolder, T. Gerlach, G. Lauer, B. Walker, J. Sullivan, R. Phillips, G. R. Pape, P. Klenerman. 2001. Sustained dysfunction of antiviral CD8+ T lymphocytes after infection with hepatitis C virus. J. Virol. 75: 5550-5558. [Abstract/Free Full Text]
  9. Appay, V., D. F. Nixon, S. M. Donahoe, G. M. A. Gillespie, T. Dong, A. King, G. S. Ogg, H. M. L. Spiegel, C. Conlon, C. A. Spina, et al 2000. HIV-specific CD8+ T cells produce antiviral cytokines but are impaired in cytolytic function. J. Exp. Med. 192: 63-75. [Abstract/Free Full Text]
  10. Moskophidis, D., F. Lechner, H. Pircher, R. M. Zinkernagel. 1993. Virus persistence in acutely infected immunocompetent mice by exhaustion of antiviral cytotoxic effector T cells. Nature 362: 758-761. [Medline]
  11. Griffin, D. E.. 2003. Immune responses to RNA-virus infections of the CNS. Nat. Rev. Immunol. 3: 493-502. [Medline]
  12. Streilein, J. W.. 1993. Immune privilege as the result of local tissue barriers and immunosuppressive microenvironments. Curr. Opin. Immunol. 5: 428-432. [Medline]
  13. González-Scarano, F., J. Martín-García. 2005. The neuropathogenesis of AIDS. Nat. Rev. Immunol. 5: 69-81. [Medline]
  14. Schneider-Schaulies, J., V. T. Meulen, S. Schneider-Schaulies. 2003. Measles infection of the central nervous system. J. Neurovirol. 9: 247-252. [Medline]
  15. Mitchell, B. M., D. C. Bloom, R. J. Cohrs, D. H. Gilden, P. G. E. Kennedy. 2003. Herpes simplex virus-1 and varicella-zoster virus latency in ganglia. J. Neurovirol. 9: 194-204. [Medline]
  16. Koralnik, I. J.. 2002. Overview of the cellular immunity against JC virus in progressive multifocal leukoencephalopathy. J. Neurovirol. 8: (Suppl. 2):59-65.
  17. McGavern, D. B., D. Homann, M. B. A. Oldstone. 2002. T cells in the central nervous system: the delicate balance between viral clearance and disease. J. Infect. Dis. 186: (Suppl. 2):S145-S151.
  18. Schweighardt, B., W. J. Atwood. 2001. Glial cells as targets of viral infection of the human nervous system. Prog. Brain Res. 132: 721-735. [Medline]
  19. Stohlman, S. A., C. C. Bergmann, S. Perlman. 1999. Mouse hepatitis virus. R. Ahmed, and I. Chen, eds. Persistent Viral Infections 537-557. Wiley, New York.
  20. Marten, N. W., S. A. Stohlman, C. C. Bergmann. 2001. MHV infection of the CNS: mechanisms of immune mediated control. Viral Immunol. 14: 1-18. [Medline]
  21. Bergmann, C. C., B. Parra, D. R. Hinton, R. Chandran, M. Morrison, S. A. Stohlman. 2003. Perforin-mediated effector function within the central nervous system requires IFN-{gamma} mediated MHC up-regulation. J. Immunol. 170: 3204-3213. [Abstract/Free Full Text]
  22. Ramakrishna, C., S. A. Stohlman, R. D. Atkinson, M. J. Shlomchik, C. C. Bergmann. 2002. Mechanisms of central nervous system viral persistence: the critical role of antibody and B cells. J. Immunol. 168: 1204-1211. [Abstract/Free Full Text]
  23. Lin, M. T., D. R. Hinton, N. W. Marten, C. C. Bergmann, S. A. Stohlman. 1999. Antibody prevents virus reactivation within the central nervous system. J. Immunol. 162: 7358-7368. [Abstract/Free Full Text]
  24. Bergmann, C. C., E. Dimacali, S. Stohl, W. Wei, M. M. C. Lai, S. Tahara, N. Marten, S. A. Stohlman. 1998. Variability of persisting MHV RNA sequences constituting immune and replication relevant domains. Virology 244: 563-572. [Medline]
  25. Ramakrishna, C., S. A. Stohlman, R. A. Atkinson, D. R. Hinton, C. C. Bergmann. 2004. Differential regulation of primary and secondary CD8+ T cells in the central nervous system. J. Immunol. 173: 6265-6273. [Abstract/Free Full Text]
  26. Hawke, S., P. G. Stevenson, S. Freeman, C. R. M. Bangham. 1998. Long term persistence of activated cytotoxic T lymphocytes after viral infection of the central nervous system. J. Exp. Med. 187: 1575-1582. [Abstract/Free Full Text]
  27. Fleming, J. O., M. Trousdale, F. El-Zaatari, S. A. Stohlman, L. P. Weiner. 1986. Pathogenicity of antigenic variants of murine coronavirus JHM selected with monoclonal antibodies. J. Virol. 58: 869-876. [Abstract/Free Full Text]
  28. González, J. M., C. C. Bergmann, B. Fuss, D. R. Hinton, C. Kangas, W. B. Macklin, S. A. Stohlman. 2005. Expression of a dominant negative IFN-{gamma} receptor on mouse oligodendrocytes. Glia 51: 22-34. [Medline]
  29. Bergmann, C. C., J. D. Altman, D. Hinton, S. A. Stohlman. 1999. Inverted immunodominance and impaired cytolytic function of CD8+ T cells during viral persistence in the central nervous system. J. Immunol. 163: 3379-3387. [Abstract/Free Full Text]
  30. Flynn, K. J., G. T. Belz, J. D. Altman, R. Ahmed, D. L. Woodland, P. C. Doherty. 1998. Virus specific CD8+ T cells in primary and secondary influenza pneumonia. Immunity 8: 683-691. [Medline]
  31. Ostler, T., T. Hussell, C. D. Surh, P. Openshaw, S. Ehl. 2001. Long-term persistence and reactivation of T cell memory in the lung of mice infected with respiratory syncytial virus. Eur. J. Immunol. 31: 2574-2582. [Medline]
  32. Chen, A. M., N. Khanna, S. A. Stohlman, C. C. Bergmann. 2005. Virus-specific and bystander CD8 T cells recruited during virus-induced encephalomyelitis. J. Virol. 79: 4700-4708. [Abstract/Free Full Text]
  33. McNally, J. M., R. M. Welsh. 2002. Bystander T cell activation and attrition. Curr. Top. Microbiol. Immunol. 263: 29-41. [Medline]
  34. McGavern, D. B., P. Truong. 2004. Rebuilding an immune-mediated central nervous system disease: weighing the pathogenicity of antigen-specific versus bystander T cells. J. Immunol. 173: 4779-4790. [Abstract/Free Full Text]
  35. Badovinac, V. P., K. A. N. Messingham, S. E. Hamilton, J. T. Harty. 2003. Regulation of CD8+ T cells undergoing primary and secondary responses to infection in the same host. J. Immunol. 170: 4933-4942. [Abstract/Free Full Text]
  36. Shen, H., J. K. Whitmire, X. Fan, D. J. Shedlock, S. M. Kaech, R. Ahmed. 2003. A specific role for B cells in the generation of CD8 T cell memory by recombinant Listeria monocytogenes. J. Immunol. 170: 1443-1451. [Abstract/Free Full Text]
  37. Früh, K., Y. Yang. 1999. Antigen presentation by MHC class I and its regulation by interferon {gamma}. Curr. Opin. Immunol. 11: 76-81. [Medline]
  38. Sedgwick, J. D., W. F. Hickey. 1997. Antigen presentation in the central nervous system. R. W. Keane, and W. F. Hickey, eds. Immunology of the Central Nervous System 364-418. Oxford University Press, Oxford.
  39. Slifka, M. K., F. Rodriguez, J. L. Whitton. 1999. Rapid on/off cycling of cytokine production by virus-specific CD8+ T cells. Nature 401: 76-79. [Medline]
  40. Redwine, J. M., M. J. Buchmeier, C. F. Evans. 2001. In vivo expression of major histocompatibility complex molecules on oligodendrocytes and neurons during viral infection. Am. J. Pathol. 159: 1219-1224. [Abstract/Free Full Text]
  41. Bergmann, C. C., B. Parra, D. R. Hinton, C. Ramakrishna, K. C. Dowdell, S. A. Stohlman. 2004. Perforin and {gamma} interferon-mediated control of coronavirus central nervous system infection by CD8 T cells in the absence of CD4 T cells. J. Virol. 78: 1739-1750. [Abstract/Free Full Text]
  42. Liu, F., J. L. Whitton. 2005. Cutting edge: re-evaluating the in vivo cytokine responses of CD8+ T cells during primary and secondary viral infections. J. Immunol. 174: 5936-5940. [Abstract/Free Full Text]
  43. Stohlman, S. A., D. R. Hinton. 2001. Viral induced demyelination. Brain Pathol. 11: 92-106. [Medline]
  44. Bachmann, M. F., R. M. Zinkernagel. 1997. Neutralizing antiviral B cell responses. Annu. Rev. Immunol. 15: 235-270. [Medline]
  45. Wong, P., E. G. Pamer. 2003. CD8 T cell responses to infectious pathogens. Annu. Rev. Immunol. 21: 29-70. [Medline]
  46. Kaech, S. M., E. J. Wherry, R. Ahmed. 2002. Effector and memory T-cell differentiation: implications for vaccine development. Nat. Rev. Immunol. 2: 251-262. [Medline]
  47. Kägi, D., F. Vignaux, B. Ledermann, K. Bürki, V. Depraetere, S. Nagata, H. Hengartner, P. Golstein. 1994. Fas and perforin pathways as major mechanisms of T cell-mediated cytotoxicity. Science 265: 528-530. [Abstract/Free Full Text]
  48. Walzer, T., A. Marcais, F. Saltel, C. Bella, P. Jurdic, J. Marvel. 2003. Cutting edge: immediate RANTES secretion by resting memory CD8 T cells following Ag stimulation. J. Immunol. 170: 1615-1619. [Abstract/Free Full Text]
  49. Cook, D. N., O. Smithies, R. M. Strieter, J. A. Frelinger, J. S. Serody. 1999. CD8 T cells are a biologically relevant source of macrophage inflammatory protein-1{alpha} in vivo. J. Immunol. 162: 5423-5428. [Abstract/Free Full Text]
  50. Biddison, W. E., D. D. Taub, W. W. Cruikshank, D. M. Center, E. W. Connor, K. Honma. 1997. Chemokine and matrix metalloproteinase secretion by myelin proteolipid protein-specific CD8+ T cells: potential roles in inflammation. J. Immunol. 158: 3046-3053. [Abstract]
  51. Olson, J. K., J. Ludovic-Croxford, S. D. Miller. 2004. Innate and adaptive immune requirements for induction of autoimmune demyelinating disease by molecular mimicry. Mol. Immunol. 40: 1103-1108. [Medline]
  52. Dai, Z., I. W. Nasr, M. Reel, S. Deng, L. Diggs, C. P. Larsen, D. M. Rothstein, F. G. Lakkis. 2005. Impaired recall of CD8 memory T cells in immunologically privileged tissue. J. Immunol. 174: 1165-1170. [Abstract/Free Full Text]
  53. Pewe, L., G. F. Wu, E. M. Barnett, R. F. Castro, S. Perlman. 1996. Cytotoxic T cell-resistant variants are selected in a virus-induced demyelinating disease. Immunity 5: 253-262. [Medline]



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