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The Journal of Immunology, 1998, 160: 2896-2904.
Copyright © 1998 by The American Association of Immunologists

CTL Effector Function Within the Central Nervous System Requires CD4+ T Cells1

Stephen A. Stohlman2,*,{dagger}, Cornelia C. Bergmann*,{dagger}, Mark T. Lin{ddagger}, Daniel J. Cua{dagger} and David R. Hinton*,{ddagger}

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


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
CTL responses induced during most viral infections are independent of help derived from the CD4+ T cell population. However, clearance of virus from the central nervous system (CNS) during infection with the neurotropic JHM strain of mouse hepatitis virus is inhibited in the absence of CD4+ T cells. Adoptive transfer of activated CD8+ T cells with virus-specific cytolytic activity into CD4+ T cell-depleted hosts demonstrated that CD4+ T cells were one component of the host response required for expression of CTL effector function(s) within the CNS. Analysis of mice infected with the JHM strain of mouse hepatitis virus demonstrated that, in contrast to CD8+ T cells, few CD4+ T cells entered the brain parenchyma. Although fewer CD8+ T cells entered the brain parenchyma in mice depleted of CD4+ T cells, access of CTL was not inhibited in the absence of CD4+ T cells. The number of apoptotic lymphocytes in the CNS increased in the absence of CD4+ T cells, suggesting that CTL enter the CNS during viral infection in a CD4-independent manner. However, these cells rapidly undergo apoptosis, indicating that expression of CTL effector function with the parenchyma of the CNS is CD4 dependent. These data raise the possibility that programmed cell death of CD8+ T cells within the CNS is due to the increased Ag present in the CNS of infected CD4 depleted mice or that autocrine cytokines, which maintain CTL activity within peripheral tissues, are inhibited in the microenvironment of the CNS.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Vigorous host immune responses are associated with recovery from acute infectious diseases and subsequent resistance to reinfection. The CD8+ CTL response predominates as the major effector arm of immunity in both clearance and protection from many viral infections (1, 2, 3). Although cytokines released from CD8+ T cells contribute to viral clearance (1, 4, 5, 6, 7), in vitro and in vivo evidence suggests that the major effector mechanism for limiting viral infection is the ability of CTL to lyse infected target cells (8, 9, 10, 11, 12, 13). It has been suggested that clearance of infections produced by noncytopathic viruses such as lymphocytic choriomeningitis virus (LCMV)3 is predominantly mediated by CTL. By contrast, clearance of cytopathic viruses is more dependent on the humoral response (2, 3). However, the roles of these effectors in the immune response to infections that localize to specific tissue sites are not well understood. Access of immune effectors is restricted from various tissues (14, 15), especially in relatively immune privileged sites such as the central nervous system (CNS). The inability of CTL to clear virus from specific tissues is readily apparent following the adoptive transfer of LCMV-specific CTL into chronic carrier mice (16, 17). CTL were relatively inefficient at reducing LCMV in brain, kidney, salivary gland, and testis, indicating that virus clearance from tissues with tight endothelium is substantially delayed. Although CTL induction and virus clearance following LCMV infection are independent of CD4+ T cells and Ab responses, clearance of persistent LCMV infection requires CD4+ T cells (18, 19, 21, 22). The mechanism(s) for this requirement is not clear; however, the role of CD4+ T cells may be to prevent exhaustion of CD8+ T cells in the presence of large amounts of Ag (3, 23), possibly via the secretion of IL-2 (4, 19, 23), which is required for both clonal expansion and prevention of apoptosis (24).

In this report we examined the requirement for CD4+ T cells in virus clearance from the parenchyma of the CNS following infection by the neurotropic JHM strain of mouse hepatitis virus (JHMV). Analysis of immune infiltrates within the CNS of rodents infected with JHMV support the importance of CTL in the antiviral response by demonstrating that CD8+ T cell accumulation coincides with declining virus titers (25, 26). The protective role of CTL during JHMV infection of the CNS was recently confirmed by the effects of adoptively transferred CTL specific for the JHMV nucleocapsid (N) protein into lethally infected recipients (27). N-specific CTL mediated protection via partial elimination of CNS virus replication. Whereas CTL suppressed virus replication in astrocytes and microglia (27), they appeared to have had little or no direct effect on virus replication in oligodendroglia.

Induction of CTL and subsequent clearance of many viruses are independent of CD4+ T cells (1, 2, 18, 28, 29). For example, vaccinia virus, influenza virus, and LCMV are cleared via a vigorous CTL response in CD4-depleted mice or in mice in which the CD4+ T cell response has been eliminated by gene disruption (29). By contrast, Rauscher leukemia virus infection and Japanese encephalitis virus infection of the CNS require both CTL and CD4+ T cells for protection (30, 31). CD8+ CTL-mediated clearance of JHMV from the CNS also appears to be dependent on CD4+ T cell-mediated help. This idea is based on the demonstration that adoptive transfer of a virus-specific CD4+ T cell population mediated protection and viral clearance from the CNS in an MHC class I-dependent manner (32). It has been further supported by the absence of JHMV clearance from mice depleted of CD4+ T cells (33) or mice in which class II has been eliminated by gene deletion (34) and the demonstrations that both virus-specific CD4+ and CD8+ T cell clones protect from lethal JHMV infections (35, 36, 37). These data suggest that the host’s immune response to CNS infection by JHMV includes a CD4+ T cell component necessary for CTL function. Whether this is an intrinsic property of a virus that exhibits cytotoxicity in vitro, but little or no direct cytopathology in vivo, or whether this is due to the relatively immune-privileged target organ of infection is not clear. In support of the idea that the site may play a role in the regulation of CTL function, excellent CTL activity can be detected in mononuclear cells isolated directly from the CNS during acute JHMV infection (38, 39, 40, 41). However, there appears to be little or no JHMV-specific CTL activity in peripheral organs during JHMV infection (38, 40, 41).

This report confirms that the ability of CTL to reduce JHMV replication in the CNS is dependent on CD4+ T cells. Mice depleted of CD4+ T cells show decreased CD8+ T cell cytolytic activity following infection, suggesting that CD4+ T cells contribute to but are not required for the development of CTL effector function. Adoptive transfers of BrdU-labeled CTL activated in vitro into infected CD4+ T cell-deficient recipients showed that entry into the CNS parenchyma was not inhibited, although the number of cells within the CNS that expressed CD8 was reduced. These data were reconciled by demonstrating the presence of increased numbers of apoptotic lymphocytes within the CNS of CD4-depleted mice. These findings indicate that CD4+ T cells are required for the maintenance of CD8+ T cell effector function within the CNS, but are not an absolute requirement for either CTL induction or trafficking of CD8+ T cells into the parenchyma during acute CNS viral infection.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

BALB/c By (H-2d), mice were obtained from The Jackson Laboratory (Bar Harbor, ME) and housed in isolator cages. Donors were immunized as previously described (27) using 1 x 106 plaque-forming units JHMV injected i.p. Recipient and control mice were infected intracranially with 100 plaque-forming units of JHMV in 30 µl of PBS. This inoculum is uniformly fatal within 7 to 9 days postinfection. Recipients were depleted of CD4+ T cells by i.p. injection of 200 µg of anti-CD4 mAb GK1.5 at -2, 0, and +2 days relative to infection as previously described (33). Mice depleted of CD8+ T cells were injected on the same schedule with 200 µg of anti-CD8 mAb 2.43. Both regimens resulted in >=98% depletion of their respective phenotypes, as analyzed by flow cytometry (see below), 4 days after the final Ab injection.

Viruses and cell lines

The DM strain, a plaque-purified isolate derived from a suckling mouse brain pool that has plaque morphology and pathogenesis consistent with those of parental JHMV (42) was propagated and plaque assayed using the murine DBT astrocytoma cell line as previously described (27, 39). Virus titers were determined by homogenization of one-half the brain in 2.0 ml of Dulbecco’s PBS, pH 7.4, using Tenbroeck tissue homogenizers. The remainder was used for histopathologic analysis (see below). Following centrifugation at 1500 x g for 10 min at 4°C, supernatants were assayed immediately or were frozen at -70°C. Virus titers were determined by plaque assay using monolayers of DBT cells as previously described (27). The data presented are the average titers of groups of at least three mice per point.

Induction and transfer of CTL effectors

Spleen cell suspensions were prepared from mice immunized 3 to 8 wk earlier. Spleen cells (1 x 108) were cultured for 6 days at 37°C in 40 ml of RPMI 1640 medium supplemented with 10% FBS (Gemini Bioproducts, Calabasas, CA), 2 mM glutamine, 25 µg/ml gentamicin, 1 mM sodium pyruvate, 5 x 10-5 M ß2-ME, and nonessential amino acids (RPMI complete) plus 10% rat Con A supernatant (RCS) containing 25 mM {alpha}-methylmannopyranoside and 1 µM pN318–335 peptide as previously described (27, 43). The 18-mer peptide containing the CTL epitope was used in most experiments due to enhanced solubility compared with that of the optimal 9-mer peptide. Viable cells were purified by centrifugation onto Lympholyte M (Accurate Chemicals, Westbury, NY) cushions and washed twice before transfer into naive recipients. Total cells (2 x 107) or CD8+ T cells (1 x 107) purified by positive selection using magnetic beads (see below) were transferred i.v. either 4 to 6 h before infection or at 2 days postinfection for BrdU-labeled cells (day 3 of infection). Recipients were challenged with JHMV within 6 h of adoptive transfer. To label CD8+ T cells for analysis of trafficking into the CNS, 50 nM BrdU (Sigma Chemical Co., St. Louis, MO) was added during in vitro expansion of memory cells for the final 48-h incubation (15). BrdU-labeled CD8+ T cells were purified by positive selection, as described below, before i.v. transfer. Inclusion of 50 nM BrdU in the culture medium for 48 h did not alter cytolytic activity compared with that in untreated cultures.

Flow cytometry and T cell purification

Cell surface expression following in vitro activation was examined using rat anti-CD4 (L3T4), rat anti-CD8 (53-5.8), rat anti-CD11a (M17/4), and rat anti-CD62L (MEL-14; PharMingen, San Diego, CA) followed by goat anti-rat FITC-F(ab')2 Ab (Caltag, South San Francisco, CA). Cells were labeled, washed, resuspended in PBS containing 0.1% formaldehyde, and analyzed on a FACStar (Becton Dickinson, Mountain View, CA). Ab plus C-mediated depletions were conducted as previously described (27), using rat anti-CD4 (mAb RL172.4) and anti-CD8 (mAb 31M).

CD8+ T cells were purified using MACS Microbeads conjugated to anti-CD8 mAb (clone 53-6.7; Miltenyi Biotec, Auburn, CA). Peptide-stimulated cells (1.5 x 108) were incubated for 40 min at 4°C with 100 µl of a colloidal microbead solution. The cell suspension was passed over a saturated steel wool column equilibrated with ice cold-PBS containing 0.5% BSA (PBS/BSA) attached to a MACS magnet (Miltenyi Biotec). The unabsorbed CD8- fraction was collected by eluting the column with 5 vol of chilled PBS/BSA at a slow flow rate regulated by a 23-gauge needle. The absorbed CD8+ fraction was collected by removing the magnet and eluting the cells with PBS/BSA. Typically, >20% of the cells applied to the column were recovered in the CD8+ fraction. Purity was >=98% as determined by flow cytometry.

CD4+ T cells from cultures stimulated in vitro with peptide pN318–335 were purified using MACS microbeads conjugated with anti-CD4 mAb (GK1.5; Miltenyi Biotec) as described above. For proliferation, CD4+ cells (1 x 105) were cultured in complete RPMI 1640 medium supplemented with 7 x 105 irradiated syngeneic spleen cells and 1% syngeneic mouse serum in 96-well plates. Cells were stimulated with lysates of UV-inactivated, JHMV-infected, DBT cells (36) or with 0.2 to 20 nM peptides (pN318–335 or pN318–326) for 72 h at 37°C and pulsed with 2 µCi of [3H]dThd (ICN Radiochemicals, Irvine, CA)/well for the last 16 h of culture. Incorporation was measured by liquid scintillation spectroscopy.

Cytotoxicity assay

Spleen cell suspensions (1 x 108) prepared from infected normal and CD4-depleted mice were cultured for 6 days at 37°C with 0.1 µM pN318–335 in 40 ml of complete RPMI 1640 medium supplemented with 10% FBS (Gemini Bioproducts). Cytolytic activity was measured in a 4-h 51Cr release assay as previously described (27, 43). J774.1 (H-2d) target cells were washed and labeled with 100 µCi of Na51CrO4 (New England Nuclear, Boston, MA). Washed target cells were preincubated for 15 min at 37°C with 0.5 to 1.0 µM peptide and added at a concentration of 1 x 104 in a 100-µl volume. Data are expressed as the percent specific release, defined as (experimental release - spontaneous release)/total (detergent release - spontaneous release). Maximum spontaneous release values were <=20% of the total release values.

Histology and cell trafficking

For histopathologic analysis mice were killed by CO2 asphyxiation. Brains were removed and bisected in the midcoronal plane. Brains and spinal cords were fixed for 3 h in Clark’s solution (75% ethanol and 25% glacial acetic acid) and embedded in paraffin. Sections were stained with either hematoxylin and eosin or luxol fast blue for routine examination. The distribution of JHMV Ag was examined using immunoperoxidase staining (Vectastain-ABC kit, Vector Laboratories, Burlingame, CA) and anti-JHMV mAb J.3.3 specific for the carboxyl terminus of the N protein (44). The distribution of BrdU+ cells was determined on paraffin-embedded sections by the immunoperoxidase method (Vectastains ABC Kit) using mouse mAb BU-33 (Sigma). To identify CD4+ and CD8+ T cells, immunoperoxidase staining was performed on acetone-fixed frozen sections using rat anti-CD4+ (L3T4, PharMingen) and rat anti-CD8+ (Ly-2, PharMingen). Anti-CD4+ and CD8+ Ab were detected with biotinylated rabbit anti-rat Ab preabsorbed with mouse serum (Vector Laboratories). Visualization was achieved using the ABC kit with 3-amino-9-ethylcarbazole as chromogen. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling was used to examine the distribution of apoptotic cells according to the supplier’s instructions (Oncor, Gaithersburg, MD).

Quantitation of lymphoid cells identified as being CD4+, CD8+, or BrdU+ was performed on immunoperoxidase-stained tissue sections counterstained with hematoxylin. Brain sections were cut in the midsagittal plane of one hemisphere and included olfactory cortex, hippocampus, and cerebellum. All positively labeled cells in the section were counted using a x40 objective, with separate computations for subarachnoid, perivascular, and parenchymal regions. Apoptotic cells were counted in a similar manner, except that the location of the cells was not differentiated since the vast majority of apoptotic cells were intraparenchymal in location.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
CD8+ T cells and viral clearance

Adoptive transfer of virus-specific CTL derived from JHMV-immunized donors expanded in vitro with either N-derived peptide pN318–335 or pN318–326 mediates MHC class I-dependent protection from an otherwise lethal infection via the reduction of JHMV replication within the CNS (27). Analysis of the transferred population by flow cytometry showed the presence of approximately equal numbers of activated (MEL-14low) CD4+ and CD11ahigh CD8+ T cells. To determine whether activated CD4+ T cells were specific for the pN318–335 peptide, CD4+ cells were purified after 6 days in vitro stimulation with peptide and tested for proliferation to the peptides containing the JHMV N-protein CTL epitope (43). No stimulation was detected using either the 18-mer pN318–335 peptide or the 9-mer pN318–326 peptide (data not shown). Minimal JHMV-specific proliferation (stimulation index = ~3) was detected, suggesting non-Ag-specific IL-2-driven in vitro expansion of the CD4+ T cells. Although an N-specific proliferative response has previously been demonstrated in BALB/c mice (45), these data indicate that neither the optimal 9-mer nor the larger 18-mer peptide contains a JHMV-specific, H-2d-restricted, CD4+ T cell epitope. However, the presence of activated CD4+ T cells suggested the possibility that this population might participate in clearance of JHMV from the CNS (35, 36, 37). To test whether the CD4+ T cells derived following in vitro activation played a role in reduction, CD8+ T cells were either depleted by mAb plus C or positively selected by magnetic bead purification and transferred to JHMV-infected recipients. Depletion of CD8+ T cells eliminated the majority, but not all, of the antiviral activity present in the CNS (Table IGo). Cells treated with C only retained the ability to reduce CNS virus replication. These data are consistent with previous reports suggesting that CD4+ T cells participate in the clearance of JHMV (35, 36, 37). By contrast, the transfer of 1 x 107 affinity-purified CD8+ T cells was as effective as transfer of 2 x 107 unfractionated cells containing approximately 50% activated CD8+ T cells (Table IGo). These data confirm that CD8+ T cells are a major effector population mediating the partial reduction of virus replication within MHC class I-positive CNS cells of JHMV-infected mice (27). Furthermore, these data indicate that the CD4+ T cells activated in vitro are not as effective as CTL at reducing virus replication within the CNS; however, CD4+ T cells activated in situ may provide protection (35, 36, 37) by an as yet unknown mechanism(s), possibly via secretion of IFN-{gamma} or Fas/Fas ligand-mediated cytolysis.


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  		Table I. CD8+ T cells mediate viral clearance from the CNS

 
CD4+ T cells are required for CTL-mediated viral clearance

The protection afforded by JHMV-specific CD4+ T cell clones requires recipients with intact immune responses (35). In vitro activated JHMV-specific CTL were therefore transferred into untreated and immunosuppressed recipients to determine whether CTL-mediated virus clearance requires participation of the host’s immune response. A single dose of irradiation was administered to recipients on the same day as the i.v. transfer of 2.5 x 107 splenocytes derived by in vitro stimulation of memory cells with pN peptide. In contrast to untreated recipients, immunosuppression prevented CTL-mediated reduction of JHMV within the CNS regardless of whether the CTL were transferred i.v. or intracranially (Table IIGo). In addition, immunosuppressed recipients were not protected from fatal disease (data not shown), similar to the prevention of CD4+ T cell-mediated protection by immunosuppression (36). The inability of CD8+ T cells to mediate complete viral clearance from the CNS (27) or protect immunosuppressed recipients (Table IIGo) suggested the participation of additional components of the host’s immune response that were activated during infection.


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  		Table II. CTL require a host immune response

 
CD4+ T cells were suggested to provide help during JHMV-specific CTL induction (32, 33); however, transfer of in vitro activated CTL was expected to obviate this requirement. To further define the role of the host’s immune response in CTL-mediated clearance, purified pN-specific CTL derived by in vitro expansion were transferred into untreated and CD4-depleted recipients. The transfer of CTL into untreated recipients reduced viral replication within the CNS and protected recipients (Table IIIGo). By contrast, no viral clearance from the CNS was detected in CTL recipients depleted of CD4+ T cells, nor were they protected from lethal disease (Table IIIGo). These data suggest that CD4+ T cells provide a critical function during JHMV infection other than providing help during CTL induction. Depletion of either CD4+ or CD8+ T cells inhibits JHMV clearance from the CNS (33), which suggested that CD4+ T cells are required during induction of JHMV-specific CTL (32, 33). To determine whether the induction of JHMV-specific CD8+ T cells is dependent on CD4+ T cells, untreated and CD4-depleted mice were infected with JHMV, and the cytolytic activity of splenocytes was determined 6 days postinfection following pN318–335 peptide restimulation in vitro. Restimulation is required as no cytolytic activity ex vivo is detectable in the spleen or cervical lymph nodes during acute JHMV infection (38, 41). CTL specific for the pN epitope were primed in the absence of CD4+ T cells (Fig. 1Go). However, 16-fold more cells were required to achieve half-maximal killing (E:T cell ratio = ~100:1) compared with cells from untreated mice (E:T cell ratio = ~6:1). The poor cytolytic activity of CTL derived from infected CD4-depleted donors was overcome by the addition of exogenous IL-2 in the form of RCS during in vitro incubation (Fig. 1Go). These data suggest that CD4+ T cells, although not required for CTL induction following JHMV infection, play a role in the development of CD8+ T cell effector function, presumably by providing help in the form of IL-2 secretion.


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  		Table III. CD4+ t cells are required for viral clearance from the CNS

 


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  		FIGURE 1. Virus-specific CTL induction in CD4-depleted, JHMV-infected, BALB/c mice. Untreated and CD4-depleted mice were infected i.c. with 100 plaque-forming units of JHMV, and 6 days postinfection, splenocytes were restimulated with 1 µM pN peptide in vitro for 6 days. The cytolytic activity of cells cultured in the absence (left panel) or the presence (right panel) of 5% RCS was tested on pN-coated J774.1 target cells at the indicated E:T cell ratios. Cytolysis is shown as the percent specific lysis of peptide-coated minus untreated J774.1 targets.

 
Entry of CD8+ T cells and maintenance of effector function

To examine the effects of CD4 and CD8 T cell depletion on the immunopathology of JHMV infection, mice were depleted of either CD4+ or CD8+ T cells as previously described (33). Both treatments were >98% effective as determined by flow cytometric analysis of splenic populations and inhibited the clearance of JHMV from the CNS (data not shown). Mice depleted of either CD4+ or CD8+ T cells showed reduced, but not absent, mononuclear cell infiltrations and increased numbers of viral Ag-positive cells compared with untreated controls (Fig. 2Go), consistent with analysis of mice rendered CD4 and CD8 deficient by gene ablation (34). These data suggested the possibility that inhibition of CTL-mediated virus clearance from the CNS in CD4-depleted recipients was not due to inhibition of CTL trafficking into the CNS. To examine this possibility, BrdU was incorporated into pN-specific CTL by incubation with 50 nM BrdU for the final 48 h of a 6-day in vitro culture (15). Positively selected CD8+ T cells were transferred to both untreated and CD4-depleted recipients at 3 days postinfection (15), and the recipients were killed 48 h later. This time point was chosen because preliminary experiments showed a significant reduction of labeled cells within the CNS >2 days after the transfer, consistent with previous results examining the trafficking of BrdU-labeled CTL into the livers of transgenic recipients expressing Ag (15). No differences in BrdU+ cells were detected in the lungs of recipients, suggesting that the efficacy of the i.v. transfers was similar in all recipients (data not shown). No difference in either the number or the distribution of BrdU+ in vitro activated CTL was detected in the CNS of JHMV-infected recipients when comparing the untreated and CD4-depleted groups (Fig. 3Go).



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  		FIGURE 2. Encephalitis and viral Ag in CD4- and CD8-depleted mice. Control mice (a and b) show perivascular lymphoid infiltrates (a, arrowheads) and scattered cells immunoreactive for viral Ag (a, arrows). At higher magnification (b), a mixed population of glial cells is positive for viral Ag. Mice depleted of either CD4+ (c and d) or CD8+ (e and f) T cells showed reduced, but not absent, mononuclear cell infiltration (c, arrow head) and increased numbers of viral Ag-positive cells (arrows) compared with untreated controls (a and b). At higher magnification (d and f), similar populations of glial cells were positive for viral Ag. Immunoperoxidase staining was performed using the avidin-biotin-peroxidase complex method for viral Ag (mAb J.3.3) with hematoxylin counterstain. Magnification, x110 (a, c, and e) and x440 (b, d, and f).

 


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  		FIGURE 3. Trafficking of BrdU-labeled CD8+ T cells in CD4-depleted recipients. BrdU was incorporated into pN-specific CTL cultures, and positively selected CD8+ T cells were transferred to both untreated (a andb) and CD4-depleted (c andd) recipients at 3 days postinfection with JHMV. Recipients were killed 48 h later. Both the number and the distribution of BrdU-labeled cells were similar in the CNS parenchyma of CD4-depleted (c and d) and control animals (a and b). The lymphoid morphology of the labeled cells is clearly seen at higher magnification (b and d). Immunoperoxidase staining was performed using the avidin-biotin-peroxidase complex method and anti-BrdU Ab with hematoxylin counterstain. Magnification, x110 (a andc) and x440 (b andd).

 
These data demonstrated that highly activated CD8+ CTL gain access to the CNS during JHMV infection independent of the presence of CD4+ T cells. Furthermore, they suggested that CTL gain access to the CNS of CD4-depleted mice, yet express little or no effector function(s). One possible mechanism to account for the apparent discrepancy is a requirement for CD4+ T cells in maintaining CTL viability. To determine whether CD4+ T cells play a role in CTL survival, the frequency and distribution of CD4+ T cells, CD8+ T cells, and apoptotic cells within the CNS of untreated and CD4-depleted CTL recipients were examined. The majority of CD4+ T cells accumulate in the perivascular and subarachnoid spaces during JHMV infection of untreated mice, with few gaining access to the CNS parenchyma (Figs. 4Go and 5a). By contrast, approximately 50% of CD8+ T cells are found within the brain parenchyma (Figs. 4Go and 5Gob). Consistent with previous results (13), few T cells are undergoing apoptosis within the CNS of JHMV-infected mice, and the majority of these are localized within the parenchyma (Fig. 5Goc). In CD4-depleted mice, no CD4+ T cells could be detected in the CNS by immunohistochemistry, consistent with the >=98% depletion of splenic CD4+ T cells following GK1.5 mAb treatment. The number of CD8+ T cells infiltrating the CNS was reduced to approximately 50% the number recruited into the CNS of mice with a normal complement of CD4+ T cells. Although the overall number of cells that stain for CD8 was reduced, the percentage of CD8+ cells within the parenchyma was approximately the same in the CD4-depleted and untreated groups (Fig. 4Go). However, the number of apoptotic cells in CD4-depleted mice increased approximately threefold (Fig. 5Go). Although a few apoptotic neurons were detected in the CD4-depleted mice, the vast majority of the cells undergoing apoptosis had the morphologic appearance of lymphocytes, consistent with the near absence of apoptotic cells in the CNS of infected, immunosuppressed mice (13). Although the identity of the apoptotic cells could not be established with certainty, morphologic characteristics, the absence of CD4+ T cells in these mice, and the predominant perivascular distribution suggest that these cells largely represent the CD8+ population. These results suggest that activated CTL traffic normally into the CNS of CD4-depleted recipients. By contrast, during infection of CD4-deficient mice, fewer CD8+ T cells were identified in the CNS. The reduction in CD8+ T cells appears to be due to increased apoptosis of this population in the absence of CD4+ T cells.



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  		FIGURE 4. Distribution of CD4+ and CD8+ T cells and apoptotic cells with the CNS of JHMV-infected mice. Cells within the perivascular and subarachnoid areas are compared with cells within the parenchyma in untreated (left panel) and CD4-depleted (right panel) mice.

 


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  		FIGURE 5. CD4+ and CD8+ T cells and apoptosis in the CNS of CD4-depleted mice. During JHMV infection of untreated mice, the majority of CD4+ T cells accumulate in the perivascular and subarachnoid spaces (a; v, vessel lumen), with few gaining access to the parenchyma. CD8+ cells are more frequently found in an intraparenchymal location (b, arrows). In control animals, few intraparenchymal T cells are undergoing apoptosis (c, arrow). The number of apoptotic cells in the CD4-depleted group (d, arrows) increased approximately threefold compared with that in the untreated mice (c). Most of the apoptotic cells are consistent in appearance with lymphoctyes (c, inset). Immunoperoxidase staining was performed using the avidin-biotin-peroxidase complex method and anti-CD4 and anti-CD8 Ab with hematoxylin counterstain. Apoptosis was detected with the terminal deoxynucleotidyl transferase-mediated, dUTP-biotin nick end-labeling method. Magnification, x110 (inset, x440).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
CD8+ CTL are major components of many antiviral immune responses. They not only reduce virus load but also correlate directly with protection from otherwise fatal outcomes (1, 2, 8, 46). The control of many viral infections by CTL is consistent with expression of their recognition elements, MHC class I molecules, on virtually all somatic cells. This contrasts with antiviral CD4+ T cell responses, which appear to be more dependent on the tropism or route of entry due to the restricted expression of MHC class II molecules on professional APC (1). Many acute infections by both cytopathic and noncytopathic viruses occur predominantly in highly vascularized tissues such as lung, spleen, and liver, in which the access of CTL does not appear to be limited by a tight endothelial cell barrier. By contrast, brain, eye, and testis are considered to be at least partially immunologically privileged, resulting in the apparent sequestration of Ag from immune recognition (14). The basis of immunologic privilege is not completely clear; however, a lack of MHC expression by specific cell types, induction of regulatory cells, expression of Fas/Fas ligand, and/or local production of cytokines that limit immunologic reactivity and tissue damage have all been suggested to play roles (47, 48, 49, 50, 51). Limited access of effectors provides an important structural barrier to a vigorous immune response to infection. Viral infections, either those limited to these organs or infections that disseminate into these organs, appear to transiently disrupt these structural barriers, allowing entry of immune effectors (15). In addition to disruption of structural barriers, expression of adhesion molecules is critical for access across these barriers and subsequent expression of effector function (52, 53). For example, during JHMV infection, CD49d is expressed on approximately 40% of CD8+ T cells obtained from the CNS parenchyma. Passive transfer of CD49d-specific mAb to infected mice inhibits both CTL-mediated virus clearance and protection (our unpublished observations). These data suggested that the CD4+ T cells might facilitate CNS access either directly by participation in the disruption of the blood-brain barrier or indirectly via an increase in expression of adhesion molecules on CNS endothelial cells. However, during the natural course of infection, CD4 cell activation may contribute to a more differentiated CD8 T cell state, potentially enhancing both cytolytic and trafficking functions. By tracking BrdU-labeled, activated, pN-specific CTL, this report demonstrates that CD4+ T cell activation is not required for the entry of activated CTL into the CNS. Although virus-specific CTL clearly influence the outcome of JHMV CNS infection (27), no antiviral CTL activity is detectable in the peripheral lymphoid organs at any time during JHMV infection (38, 39, 40, 41). One interpretation of these data is that the JHMV-specific CD8+ T cells that express perforin-mediated cytolytic activity are either expanded within the CNS and/or mature into lytic CTL within the microenvironment of the CNS (38). The extent of CD4 T cell involvement is unknown. Comparison of mononuclear cell infiltrates in the CNS of JHMV-infected untreated and CD4-depleted mice showed that, in contrast to the adoptive transfer of activated CTL, the number of CD8+ T cells within the parenchyma was significantly reduced in the absence of CD4+ T cells. These data are consistent with the idea that CD4+ T cells support the entry of CD8+ T cell into the CNS parenchyma from the perivascular or subarachnoid spaces. However, a dramatic increase in the number of apoptotic cells was noted in infected CD4-depleted mice. We have been unable to visualize surface markers on BrdU-labeled apoptotic cells by immunohistochemistry, which may be due to decreased CD8 expression on Ag-reactive cells (54). However, these data suggest that CD8+ T cells that express CTL activity in vitro enter the CNS in a CD4+ T cell-independent manner, similar to the ability of CTL to enter other highly vascularized tissues (15, 16, 17). In contrast to the maintenance of CTL effector function in peripheral tissues (15, 16, 17, 29), the effector function of CTL within the CNS parenchyma of the CD4-depleted, JHMV-infected mice appears compromised as measured by their inability to clear virus from the CNS. This loss of function appears to correlate with increased apoptosis in the absence of CD4+ T cells.

Whether the increased apoptosis is due to the absence of a tropic factor(s) required for CTL survival or increased Ag load within the CNS of CD4-depleted mice is currently under investigation. However, reduced CTL activity in splenocytes from CD4-depleted infected mice and the recovery of cytolytic activity by IL-2 support its role in enhancing CTL survival and optimal induction of effector function (4, 23, 24). These data suggest that increased apoptotic lymphocytes within the CNS of CD4-depleted recipients may reflect depletion of cytokines or growth factors provided by CD4+ T cells. Indeed, CTL induction is independent of IL-2 (23); however, expression of cytolytic activity is supported by an autocrine loop of IL-2 secreted by activated CD8+ T cells (18, 19, 23). Our data support the idea that CD8+ T cells within the CNS parenchyma may be unable to induce or maintain lytic function due to a defect in the autocrine IL-2 secretion supplemented by the CD4+ T cell population. However, the present data cannot exclude the possibility that the effect on CTL activity is indirect and derived from other cytokines or is a factor(s) derived from endogenous CNS cells (46, 51, 55). For example, CD4+ T cells may secrete or induce the secretion of anti-inflammatory cytokines, such as IL-4, IL-7, IL-10, and IL-15, which maintain or augment CTL activity (55, 56, 57, 58, 59). Both IL-4 and IL-10 mRNA levels increase within the CNS of JHMV-infected mice at the time of viral clearance, although very little IL-4 mRNA can be detected (60). In this regard it is interesting that virus replicates to a higher titer in the CNS of mice rendered deficient in IL-10 by homologous recombination compared with that in control mice (our unpublished observations). Although this may suggest that IL-10 contributes to the CTL activity in the CNS, IL-10 is secreted from CD4- endogenous CNS cells during inflammatory responses (56, 61), and few Th2 type CD4+ T cells are present within the CNS of JHMV-infected mice, as judged by the absent (62) or low (60) levels of IL-4 mRNA detected.

JHMV infection of cells in vitro results in cytopathic effects characterized by giant cell formation. However, in vivo there are few giant cells or apoptotic cells in the absence of an immune response. This suggests that in vivo JHMV infection may be more closely related to infections by noncytopathic viruses, such as LCMV and HIV. Analysis of mice infected with high doses of LCMV suggested that CD4+ T cells may be important in preventing CTL exhaustion due to a high Ag load (2, 3, 22, 23). However, this interpretation is controversial, since other data suggest that CD4+ T cells are required to sustain CTL activity and CTL memory during the high Ag load associated with persistent LCMV infection (18, 21, 22, 63). Whether the requirement for CD4+ T cells is related to their ability to assist in the reduction of viral load during JHMV infection via secretion of antiviral cytokines or the provision of B cell help is not clear. However, CD4+ T cells may also contribute directly to the prevention of CTL exhaustion by the provision of cytokines, such as IL-2. In contrast to JHMV infection of the CNS, the reduction in LCMV-specific CTL activity does not occur until after acute infection, and neither CTL-mediated viral clearance or protection from acute infection is compromised via elimination of CD4+ T cells (18, 21, 22, 29, 63). The JHMV Ag load within the CNS appears sufficient for CD8+ T cells to overcome a requirement for CD4-derived IL-2 during induction (4, 19); however, cytokine secreted by activated CD4+ T cells may be required to maintain the lytic activity or viability of virus-specific CTL within the CNS. In addition to the requirement(s) for CD4+ T cells to secrete cytokines, the ability to inhibit virus replication and mediate protection from JHMV infection may also be attributed to differences in the access of CD8+ T cells to peripheral tissues vs the CNS parenchyma (15).

In the present report the host immune response assisting CTL-mediated clearance of JHMV from the CNS was dependent on CD4+ T cells. The data show that the CTL response induced by JHMV infection in the absence of CD4+ T cells is reduced, but not abrogated, compared with that in normal mice. Therefore, JHMV infection, although restricted to the CNS, is similar to the majority of other viruses examined (1) in that CTL are initially induced in a CD4+ T cell-independent manner. However, in determining the basis for the requirement for CD4+ T cells in the antiviral activity of CTL within the CNS, the data show that activated CTL can access the CNS in the absence of CD4+ T cells. These CTL appear to be nonfunctional, since there is little or no evidence for CTL-mediated virus clearance in CD4+ T cell-depleted mice even following the adoptive transfer of highly activated CTL. This interpretation is consistent with histologic analysis of the CNS of mice depleted of CD4+ T cells, which exhibits a reduction in mononuclear cell infiltration, yet an increase in the number of virus-infected cells. These data suggest that the CNS differs in a fundamental aspect from peripheral organs, which allow ready access to activated CTL that appear to be able to recognize Ag-bearing target cells and clear virus (15).


   Acknowledgments
 
The authors appreciate the excellent technical assistance of Wenqiang Wei, Qin Yao, and Chung Kang Ho.


   Footnotes
 
1 This work was supported by U.S. Public Health Service Grants NS18146 and AI33314 and by U.S. Public Health Service Training Grant NS7149 (to D.C.). Back

2 Address correspondence and reprint requests to Dr. S. Stohlman, University of Southern California, MCH 142, 1333 San Pablo St., Los Angeles, CA 90033. Back

3 Abbreviations used in this paper: LCMV, lymphocytic choriomeningitis virus; CNS, central nervous system; JHMV, JHM strain of mouse hepatitis virus; N, nucleocapsid; BrdU, bromodeoxyuridine; RCS, rat concanavalin A supernatant; low (superscript), low level; high (superscript), high level. Back

Received for publication August 20, 1997. Accepted for publication November 19, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Koszinowski, U. H., M. J. Reddehase, S. Jonjic. 1991. The role of CD4 and CD8 T cells in viral infections. Curr. Opin. Immunol. 3:471.[Medline]
  2. Kagi, D., H. Hengartner. 1996. Different roles for cytotoxic T cells in the control of infections with cytopathic versus noncytopathic viruses. Curr. Opin. Immunol. 8:472.[Medline]
  3. Zinkernagel, R. M.. 1996. Immunology taught by viruses. Science 271:173.[Abstract]
  4. Biron, C. A.. 1994. Cytokines in the generation of immune responses to, and resolution of, virus infection. Curr. Opin. Immunol. 6:530.[Medline]
  5. Ruby, J., I. Ramshaw. 1991. The antiviral activity of immune CD8+ T cells is dependent on interferon-g. Lymphokine Cytokine Res. 10:353.[Medline]
  6. Kundig, T. M., H. Hengartner, R. M. Zinkernagel. 1993. T cell-dependent IFN-{gamma} exerts an antiviral effect in the central nervous system but not in peripheral solid organs. J. Immunol. 150:2316.[Abstract]
  7. Ramsay, A., J. Ruby, I. A. Ramshaw. 1993. A case for cytokines as effector molecules in the resolution of virus infection. Immunol. Today 14:155.[Medline]
  8. Zinkernagel, R. M., E. Haenseler, T. Leist, A. Cerny, Hengartner H., A. Althage. 1986. T cell-mediated hepatitis in mice infected with lymphocytic choriomeningitis virus: liver cell destruction by H-2 class I-restricted virus-specific cytotoxic T cells as a physiological correlate of the 51Cr-release assay?. J. Exp. Med. 164:1075.[Abstract/Free Full Text]
  9. Castelmur, I., C. DiPaolo, M. F. Bachmann, H. Hengartner, R. M. Zinkernagel, T. M. Kundig. 1993. Comparison of the sensitivity of in vivo and in vitro assays for detection of antiviral cytotoxic T cell activity. Cell. Immunol. 151:460.[Medline]
  10. Ando, K., L. G. Guidotti, S. Wirth, T. Ishikawa, G. Missale, T. Moriyama, R. D. Schreiber, H.-J. Schlicht, S. Huang, F. V. Chisari. 1994. Class I-restricted cytotoxic T lymphocytes are directly cytopathic for their target cells in vivo. J. Immunol. 152:3245.[Abstract]
  11. Walsh, C. M., M. Matloubian. C.-C. Liu, R. Ueda, C. G. Kurahara, J. L. Christensen, M. T. F. Huang, J. D.-E. Young, R. Ahmed, W. R. Clark. 1994. Immune function in mice lacking the perforin gene. Proc. Natl. Acad. Sci. USA 91:10854.[Abstract/Free Full Text]
  12. Kagi, D., B. Ledermann, K. Burki, P. Seiler, B. Odermatt, K. J. Olsen, E. R. Podack, R. M. Zinkernagel, H. Hengartner. 1994. Cytotoxicity mediated by T cells and natural killer cells is greatly impaired in perforin-deficient mice. Nature 369:31.[Medline]
  13. Lin, M. T., S. A. Stohlman, D. R. Hinton. 1997. Mouse hepatitis virus is cleared from the central nervous systems of mice lacking performin-mediated cytolysis. J. Virol. 71:383.[Abstract]
  14. Barker, C. F., P. F. Bartlett. 1977. Immunologically privileged sites. Adv. Immunol. 25:1.[Medline]
  15. Ando, K., L. G. Guidotti, A. Cerny, T. Ishikawa, F. V. Chisari. 1994. CTL access to tissue antigen is restricted in vivo. J. Immunol. 153:482.[Abstract]
  16. Oldstone, M. B. A., P. Blount, P. J. Southern. 1986. Cytoimmunotherapy for persistent virus infection reveals a unique clearance pattern from the central nervous system. Nature 321:239.[Medline]
  17. Ahmed, R., B. D. Jamieson, D. D. Porter. 1987. Immune therapy of a persistent and disseminated viral infection. J. Virol. 61:3920.[Abstract/Free Full Text]
  18. Ahmed, R., L. D. Butler, L. Bhatti. 1988. T4+ T helper cell function in vivo: differential requirement for induction of antiviral cytotoxic T-cell and antibody responses. J. Virol. 62:2102.[Abstract/Free Full Text]
  19. Kasaian, M. T., K. A. Leite-Morris, C. A. Biron. 1991. The role of CD4+ cells in sustaining lymphocyte proliferation during lymphocytic choriomeningitis virus infection. J. Immunol. 146:1955.[Abstract]
  20. Jamieson, B. D., L. D. Butler, R. Ahmed. 1987. Effective clearance of a persistent viral infection requires cooperation between virus-specific Lyt2+ T cells and nonspecific bone marrow-derived cells. J. Virol. 61:3930.[Abstract/Free Full Text]
  21. von Herrath, M., M. Yokoyama, J. Dockter, M. B. A. Olstone, J. L. Whitton. 1996. CD4-deficient mice have reduced levels of memory CTL after immunization and show diminished resistance to subsequent virus challenge. J. Virol. 70:1072.[Abstract]
  22. Moskophidis, D., F. Lechner, H. P. Pircher, R. M. Zinkernagel. 1993. Virus persistence in acutely infected immunocompetent mice by exhaustion of anti-viral CTL. Nature 362:758.[Medline]
  23. Cohen, J. J.. 1993. Apoptosis. Immunol. Today 14:126.[Medline]
  24. Williamson, J. S.-P., K. Sykes, S. A. Stohlman. 1991. Characterization of brain infiltrating mononuclear cells during infection with mouse hepatitis virus strain JHM. J. Neuroimmunol. 32:199.[Medline]
  25. Dorries, R., S. Schwender, H. Imrich, H. Harms. 1991. Population dynamics of lymphocyte subsets in the central nervous system of rats with different susceptibility to coronavirus-induced demyelinating encephalitis. Immunology 74:539.[Medline]
  26. Stohlman, S. A., C. C. Bergmann, R. C. van der Veen, D. R. Hinton. 1995. Mouse hepatitis virus-specific cytotoxic T lymphocytes protect from lethal infection without eliminating virus from the central nervous system. J. Virol. 69:684.[Abstract]
  27. Leist, T. P., S. P. Cobbold, H. Waldmann, M. Aguet, R. M. Zinkernagel. 1987. Functional analysis of T lymphocyte subsets in antiviral host defense. J. Immunol. 138:2278.[Abstract]
  28. Doherty, P. C.. 1993. Virus infections in mice with targeted gene disruptions. Curr. Opin. Immunol. 5:479.[Medline]
  29. Hom, R. C., R. W. Finberg, S. Mullaney, R. M. Ruprecht. 1991. Protective cellular retroviral immunity requires both CD4+ and CD8+ immune T cells. J. Virol. 65:220.[Abstract/Free Full Text]
  30. Murali-Krishna, K., V. Ravi, R. Munjanath. 1996. Protection of adult but not newborn mice against lethal intracerebral challenge with Japanese encephalitis virus by adoptively transferred virus-specific cytotoxic T lymphocytes: requirement for L3T4+ T cells. J. Gen. Virol. 77:705.[Abstract/Free Full Text]
  31. Sussman, M. A., R. A. Shubin, S. Kyuwa, S. A. Stohlman. 1989. T-cell-mediated clearance of mouse hepatitis virus strain JHM from the central nervous system. J. Virol. 63:3051.[Abstract/Free Full Text]
  32. Williamson, J. S.-P., S. A. Stohlman. 1990. Effective clearance of mouse hepatitis virus from the central nervous system requires both CD4+ and CD8+ T cells. J. Virol. 64:4589.[Abstract/Free Full Text]
  33. Houtman, J. J., J. O. Fleming. 1996. Dissociation of demyelination and viral clearance in congenitally immunodeficient mice infected with murine coronavirus JHM. J. Neurovirol. 2:101.[Medline]
  34. Stohlman, S. A., G. K. Matsushima, N. Casteel, L. P. Weiner. 1986. In vivo effects of coronavirus-specific T cell clones: DTH inducer cells prevent a lethal infection but do not inhibit virus replication. J. Immunol. 136:3052.[Abstract]
  35. Yamaguchi, K., N. Goto, S. Kyuwa, M. Hayami, Y. Toyoda. 1991. Protection of mice from a lethal coronavirus infection in the central nervous system adoptive transfer of virus-specific T cell clones. J. Neuroimmunol. 32:1.[Medline]
  36. Korner, H., A. Schliephake, J. Winter, F. Zimprich, H. Lassmann, J. Sedgwick, S. Siddell, H. Wege. 1991. Nucleocapsid or spike protein-specific CD4+ T lymphocytes protect against coronavirus-induced encephalomyelitis in the absence of CD8+ T cells. J. Immunol. 147:2317.[Abstract]
  37. Stohlman, S. A., S. Kyuwa, J. Polo, D. Brady, M. M. C. Lai, C. C. Bergmann. 1993. Characterization of mouse hepatitis virus specific cytotoxic T cells derived from the central nervous system of mice infected with the JHM strain. J. Virol. 67:7050.[Abstract/Free Full Text]
  38. Castro, R. F., G. D. Evans, A. Jaszewski, S. Perlman. 1994. Coronavirus-induced demyelination occurs in the presence of virus-specific cytotoxic T cells. Virology 200:733.[Medline]
  39. Castro, R. F., S. Perlman. 1995. CD8+ T cell epitopes within the surface glycoprotein of a neurotropic coronavirus and correlation with pathogenicity. J. Virol. 69:8127.[Abstract]
  40. Castro, R. F., S. Perlman. 1996. Differential antigen recognition by T cells from the spleen and central nervous system of coronavirus-infected mice. Virology 222:247.[Medline]
  41. Stohlman, S. A., P. R. Braton, J. O. Fleming, L. P. Weiner, M. M. C Lai. 1982. Murine coronaviruses: isolation and characterization of two plaque morphology variants of the JHM neurotropic strain. J. Gen. Virol. 63:265.[Abstract/Free Full Text]
  42. Bergmann, C., M. McMillan, S. A. Stohlman. 1993. Characterization of the Ld restricted CTL epitope in the mouse hepatitis virus nucleocapsid protein. J. Virol. 67:7041.[Abstract/Free Full Text]
  43. Stohman, S. A., C. Bergmann, D. Cua, H. Wege, R. van der Veen. 1994. Location of antibody epitopes within the mouse hepatitis virus nucleocapsid protein. Virology 202:146.[Medline]
  44. van der Veen, R. C.. 1996. Immunogenicity of JHM virus proteins: characterization of a CD4+ T cell epitope on nucleocapsid protein which induces different T-helper cell subsets. Virology 225:339.[Medline]
  45. Sykulev, Y., M. Joo, I. Vturina, T. Tsomides, H. Eisen. 1996. Evidence that a single peptide/MHC complex on a target cell can elicit a CTL response. Immunity 4:565.[Medline]
  46. Irani, D. N., K.-I. Lin, D. E. Griffin. 1997. Regulation of brain-derived T cells during acute central nervous system inflammation. J. Immunol. 158:2318.[Abstract]
  47. Griffith, T. S., T. Brunner, S. M. Fletcher, D. R. Green, T. A. Ferguson. 1995. Fas ligand-induced apoptosis as a mechanism of immune privilege. Science 270:1189.[Abstract/Free Full Text]
  48. Foster, C. S., J. G. Monroe, R. Campbell, J. Kalpaxis, R. Wetzig, M. I. Green. 1968. Ocular immune responses. II: Priming of A/J mice in the vitreous induces either enhancement or suppression of subsequent hapten-specific DTH responses. J. Immunol. 136:2787.[Abstract]
  49. Strelein, J. W.. 1995. Unraveling immune privilege. Science 270:1158.[Abstract/Free Full Text]
  50. Irani, D. N., D. E. Griffin. 1996. Regulation of lymphocyte homing into the brain during viral encephalitis at various stages of infection. J. Immunol. 156:3850.[Abstract]
  51. Christensen, J. P., E. C. Andersson, A. Scheynius, O. Marker, A. R. Thomsen. 1995. {alpha}4 integrin directs virus-activated CD8+ T cells to sites of infection. J. Immunol. 154:5293.[Abstract]
  52. Rodrigues, M., R. S. Nussenzweig, P. Romero, F. Zavala. 1992. The in vivo cytotoxic activity of CD8+ T cell clones correlate with their levels of expression of adhesion molecules. J. Exp. Med. 175:895.[Abstract/Free Full Text]
  53. Zhang, L., W. Fung-Leung, R. G. Miller. 1995. Down-regulation of CD8 on mature antigen-reactive T cells as a mechanism of peripheral tolerance. J. Immunol. 155:3464.[Abstract]
  54. Akbar, A. N., N. J. Borthwick, R. G. Wickremasinghe, P. Panayoitidis, D. Pilling, M. Bofill, S. Krajewski, J. C. Reed, M. Salmon. 1996. Interleukin-2 receptor common {gamma}-chain signaling cytokines regulate activated T cell apoptosis in response to growth factor withdrawal: selective induction of anti-apoptotic (bcl-2, bcl-xL) ut not pro-apoptotic (bax, bcl-xS) gene expression. Eur. J. Immunol. 26:294.[Medline]
  55. Benveniste, E. N.. 1992. Inflammatory cytokines within the central nervous system: sources, function and mechanism of action. Am. J. Physiol. Cell Physiol. 263:32.
  56. Widmer, M. B., K. H. Grabstein. 1987. Regulation of cytolytic T-lymphocyte generation by B-cell stimulatory factor. Nature 326:795.[Medline]
  57. Pfeifer, J. D., D. T. McKenzie, S. L. Swain, R. W. Dutton. 1987. B cell stimulatory factor 1 (interleukin 4) is sufficient for the proliferation and differentiation of lectin-stimulated cytolytic T lymphocyte precursors. J. Exp. Med. 166:1464.[Abstract/Free Full Text]
  58. Chen, W.-F., A. Zlotnik. 1991. Interleukin 10: a novel cytotoxic T cell differentiation factor. J. Immunol. 147:528.[Abstract]
  59. Parra, B., D. R. Hinton, M. T. Lin, D. J. Cua, S. A. Stohlman. 1997. Kinetics of cytokine mRNA expression in the central nervous system following lethal and nonlethal coronavirus-induced acute encephalomyelitis. Virology 233:260.[Medline]
  60. Kennedy, M. K., D. S. Torrance, K. S. Picha, K. M. Mohler. 1992. Analysis of cytokine mRNA expression in the central nervous system of mice with experimental autoimmune encephalomyelitis reveals that IL-10 mRNA expression correlates with recovery. J. Immunol. 149:2496.[Abstract]
  61. Pearce, B. D., M. V. Hobbs, T. S. McGraw, M. Buckmeier. 1994. Cytoline induction during T-cell mediated clearance of mouse hepatitis virus from neurons in vivo. J. Virol. 68:5483.[Abstract/Free Full Text]
  62. Matloubian, M., R. J. Concepcion, R. Ahmed. 1994. CD4+ T cells are required to sustain CD8+ T-cell responses during chronic viral infection. J. Virol. 68:8056.[Abstract/Free Full Text]



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