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The Journal of Immunology, 2007, 178: 5812-5819.
Copyright © 2007 by The American Association of Immunologists, Inc.
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Enrichment and Persistence of Virus-Specific CTL in the Brain of Simian Immunodeficiency Virus-Infected Monkeys Is Associated with a Unique Cytokine Environment1

Maria Cecilia G. Marcondes2,*, Tricia H. Burdo*, Sieghart Sopper{dagger}, Salvador Huitron-Resendiz*, Caroline Lanigan*, Debbie Watry*, Claudia Flynn*, Michelle Zandonatti* and Howard S. Fox*

* Molecular and Integrative Neurosciences Department, The Scripps Research Institute, La Jolla, CA 92037; and {dagger} Department of Virology and Immunology, Deutsches Primatenzentrum, Göttingen, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The host reaction to infection of the brain contributes to a number of CNS pathologies including neuro-AIDS. In this study, we have identified the accumulation of SIV-specific CTL in the brains of SIV-infected animals who have neurophysiological abnormalities but are otherwise asymptomatic. SIV-specific CTL enter the brain early after viral infection and are maintained in the brain even when those reactive with an immunodominant epitope in Tat are lost from the rest of the body. The specialized CNS environment contributes to this unique outcome. Following SIV infection, brain levels of IL-15 were significantly elevated whereas IL-2 was absent, creating an environment that favors CTL persistence. Furthermore, in response to IL-15, brain-derived CD8+ T cells could expand in greater numbers than those from spleen. The accumulation, persistence, and maintenance of CTL in the brain are closely linked to the increased levels of IL-15 in the absence of IL-2 in the CNS following SIV infection.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The presence of basal levels of T cells in the normal brain is important as a mechanism of immune surveillance. However, the accumulation of T cells in the brain is potentially harmful, as only activated cells, which have proinflammatory properties, cross the blood-brain barrier (1). CTL, which bear the CD8 coreceptor for the Ag-class I MHC, are critical in eliminating or controlling viral infection. However, in the brain their activity can also be harmful by killing crucial cells either directly or indirectly through bystander damage.

During HIV infection of people and SIV infection of monkeys, CTL are present in the CNS early after infection (2, 3, 4). Experiments in SIV-inoculated monkeys reveal that virus enters the brain early (5). CD8+ T cells isolated at early time points have altered phenotypes relative to those from uninfected brains, in that they now express CTL effector molecules (6). The brain viral load then decreases by 10- to a 1000-fold, but the CD8+ T cells increase in number in the brain and persist in this phase of infection (6, 7). The relationship between the CNS CTL response and that in the periphery is unknown. Furthermore, although this chronic phase is relatively asymptomatic both in the brain and periphery, neurophysiological testing consistently reveals signs of CNS dysfunction, perhaps due to this ongoing CNS immune response (6, 7, 8).

T cells that accumulate in the brain may recognize their cognate Ags (9), however, additional survival signals provided by the tissue are likely necessary. Some cytokines, such as IL-2, IL-15, and IL-7, can provide survival or proliferation signals to CD8 cells (10). Their presence may help shape a unique pattern of CTL specificities in the brain vs the rest of the body. Therefore, in this study, we have examined the epitope specificity of CD8 cells in the brain and changes on cytokines that can affect CTL survival. Our study offers an explanation for accumulation and persistency of CTLs that enter early in the brain in viral neuropathology and, perhaps, in other immune-mediated CNS diseases.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Animals and infection

STLV, SRV-type D, SIV, and Cercopithecine herpesvirus-1 free rhesus macaques were purchased from Labs of Virginia (Yemassee, SC) and infected i.v. with a cell-free stock of SIVmac182, using 1.25 ng of p27Gag/animal. All animal experiments were performed as previously described (6) with approval from The Scripps Research Institute Institutional Animal Care and Use Committee.

Viral quantitation

SIV RNA in plasma, cerebrospinal fluid; tissue was determined using the quantitative branched DNA signal amplification assay performed by the Bayer Reference Testing Laboratory (Emeryville, CA).

Electrophysiology

Electrophysiological analysis of brainstem auditory evoked potentials was performed on ketamine (20 mg/kg) anesthetized animals as described previously (8).

Cells

Buffy coats obtained from centrifugation of EDTA-anticoagulated blood, centrifuged cells recovered from bronchioalveolar lavage (BAL),3 and cell suspensions recovered from spleen, deep cervical and inguinal lymph nodes, and liver obtained from pressing organs through a nylon mesh, were submitted to a Ficoll-Isopaque (Pharmacia Biotech) gradient centrifugation for isolation of the mononuclear fraction. Cells from brain were isolated as previously described (6).

Cell cultures

Cryopreserved splenic or brain-derived cell suspensions were thawed and resuspended in RPMI 1640 10% FBS. Cells were cultured at a density of 4 x 106 cells/well in 48-well plates, with or without different concentrations of rIL-2 or IL-15 (BioSource International). Media (with replenished cytokine) were changed every at 3 days and cells were harvested at day 7.

Flow cytometry

Cells were surface stained with mixtures of Abs in staining buffer (HBSS with 2% FCS and 0.01% NaN3). The cell surface Abs used for the staining were previously described (6) with the addition of PE-labeled TatSL8 and GagCM9 tetramers (Beckman Coulter). The level of tetramer-positive cells was determined as the percentage of gated CD3+CD8+ lymphocytes. Intracellular Ki67 detection was performed using FITC-labeled Ki67 (B56; BD Biosciences) on cells that were briefly washed with BD FACS Lysing Solution (BD Biosciences), fixed with 4% paraformaldehyde for 20 min, and permeabilized using 0.3% Triton X-100 in PBS containing 2% FBS and 0.2% sodium azide. Flow cytometric data were acquired using a FACSCalibur flow cytometer (BD Biosciences) and analysis was performed with FlowJo 6.2.1 software (Tree Star).

Quantitative RT-PCR

RNA was isolated, purified, quantified, and used as a template for cDNA as previously described (11), with the addition of a phenol extraction and Qiagen RNeasy column purification for the liver RNA. Real-time PCR was performed using gene-specific primers and probes (Table I). A dilutional analysis of the samples indicated a correlation coefficient for concentration and cycle threshold (Ct) of 0.99. Reactions were performed on a MX3000 (Stratagene) using Supermix UDG (Invitrogen Life Technologies). The delta Ct (dCt) method was performed to determine relative concentrations, using the average of the Ct of 18S, GAPDH, and TATA box-binding protein (TBP) as the normalizing value. Relative units were calculated as 2dCt.


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  		Table I. Sequence of primers and probes used for real-time PCRa

 
Viral sequencing

Plasma was ultracentrifuged at 33,000 rpm (Beckman Coulter SW 50.1 rotor) for 35 min to pellet virus, followed by use of the Totally RNA kit (Ambion) for RNA isolation. cDNA from plasma as well as brain RNA was synthesized using the first-strand cDNA synthesis system (Marligen Biosciences) and the genomic region corresponding to the first exon of Tat was amplified using Titanium Taq (BD Biosciences) and primers based on prior sequencing of related viruses (Table I). The PCR products were ligated and transformed using the TOPO TA cloning kit (Invitrogen Life Technologies). Positive clones were sequenced on both strands by a commercial service (Retrogen).

Immunohistochemistry

Indirect immunohistochemical staining was performed on formalin-fixed, paraffin-embedded tissue sections using Ag retrieval as previously described (7), using dilutions and Ag recovery methods optimized for the Ag/Ab. Trypsin treatment was used for the detection of IL-15 (goat polyclonal; R&D Systems), citrate treatment for glial fibrillary acidic protein (GFAP, rabbit polyclonal; Zymed Laboratories), and tris/urea treatment for CD8 (rabbit polyclonal Ab; Panomics).

Statistical analysis

Group comparisons were performed using the tests described in the text and figure legends. The difference between the means was considered significant at {alpha} < 0.05. Tests were performed using the software packages StatGraphics Plus 5.0 (StatPoint) and Prism (GraphPad Software).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Longitudinal blood and CsF (3) CTL SIV epitope specificity

In four SIV-infected, MamuA*01+ animals, the CD8+ T cell response against SIV was analyzed by tetramer staining and FACS analysis, assessing reactivity against the two major immunodominant viral epitopes found in the acute phase, GagCM9 and TatSL8 (12). In PBMC, as reported by others (12, 13, 14), the anti-TatSL8 response appeared early and then largely disappeared (Fig. 1A). The anti-GagCM9 response, in contrast, also appeared early, but was maintained at a relatively constant level throughout the period of observation (Fig. 1B). The CNS was sequentially sampled through examination of the cerebrospinal fluid. Both TatSL8- and GagCM9-reactive cells appeared early after infection (Fig. 1, C and D). Similar to the blood, TatSL8-reactive cells then declined, but in contrast to the blood, remained detectable throughout the observation period. The GagCM9-reactive cells increased in abundance in the early postinfection (p.i.) period and then remained at constant levels. Interestingly, for both epitopes, tetramer-reactive cells were significantly enriched in the cerebrospinal fluid in comparison to the blood. Furthermore, in the CsF, the tetramer-reactive cells comprised a relatively high percentage (10–20%) of all CD8+ T cells, as opposed to 1–7% in blood.


Figure 1
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  		FIGURE 1. Kinetics of TatSL8 and GagCM9-specific CD8+ T cells in the blood (A and B) and CsF (C and D) along SIV infection. Note the differences in y-axis scale for blood (A and B) and cerebrospinal fluid (C and D). Contemporaneous studies in an uninfected control MamuA*01+ monkey (animal 408, before its subsequent inoculation with SIV) never exceeded tetramer proportions of 0.08% for blood and 0.2% for cerebrospinal fluid. Individual animals is indicated by regular lines; the mean of the four animals is indicated by a bold line. Two-way repeated measures ANOVA indicates that for both TatSL8 and GagCM9, cerebrospinal fluid levels were greater than those in blood (p = 0.0126 and p = 0.0175, respectively). For TatSL8, a significant effect was also found for time p.i. (p < 0.0001). One-way repeated measures ANOVA with post hoc Tukey’s test revealed that in the blood, TatSL8-reactive cells were higher at day 28 p.i. than all other time points (ANOVA p < 0.001, Tukey’s test p < 0.01), whereas in the cerebrospinal fluid TatSL8-reactive cells were higher at day 28 than the day 59, 77, and 154 time points (ANOVA p = 0.0154, Tukey’s test p < 0.05).

 
Tissue CTL specificity

At ~6 mo after inoculation, the animals were in a chronic phase of infection with stable plasma and CsF viral loads and were clinically asymptomatic (although signs of CNS dysfunction were present by neurophysiological testing, Fig. 2). They were sacrificed and perfused to eliminate blood-borne cells from the organs. As opposed to the lymphoid organs, in the blood, liver, BAL, CsF, and brain, CD8+ T cells were more abundant than CD4+ T cells. In the brain, when compared with uninfected monkeys, there was a significant (5.6-fold, p = 0.002, two-tailed t test) increase in the number of CD8+ T cells, consistent with our previous findings (3, 6, 7).


Figure 2
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  		FIGURE 2. Longitudinal course of infection. A, Plasma (filled symbols) and CsF (open symbols) viral load at indicated time points after viral inoculation (mean ± SEM). One-way repeated measures ANOVA reveals that both plasma and cerebrospinal fluid viral loads vary significantly over time (p < 0.0001 for both), with Tukey’s post hoc test revealing that day 14 is significantly higher than the other days in both fluids (p < 0.001), except that in the cerebrospinal fluid day 28 is not distinguishable from day 14. B, Latency of the P3 wave of the brainstem auditory evoked potential. Time 0 is the average of four preinoculation determinations (±SD). Postinoculation values for given as the mean (±SEM). One-way repeated measures ANOVA with post hoc Dunnett’s test indicate that day 77 onward are significantly greater than the baseline uninfected value (ANOVA p = 0.0160, Dunnett’s test p < 0.01).

 
The proportion of CD8+ T cells specific for the immunodominant TatSL8 and GagCM9 epitopes was then analyzed in the different organs. Similar to the findings in blood, the frequency of TatSL8-specific cells was low to undetectable in other peripheral sites and, as found above, at moderate levels in the CsF (Fig. 3). Surprisingly, the TatSL8-specific CD8+ T cells were at high levels in the brain, comprising an average of 15% of these cells. GagCM9-specific cells were present throughout the body (Fig. 3), but were present at the highest level in the brain, averaging 22% of the CD8+ T cells, similar to the findings in vaccinated, SIV-infected rhesus monkeys (15).


Figure 3
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  		FIGURE 3. Proportion of CD8+ T cells specific for the immunodominant epitopes within Tat (open bars) and Gag (filled bars) within the CD3+CD8+ gate in the indicated sites in the four individual animals.

 
The presence of a high proportion of TatSL8-specific cells at 6 mo p.i. in the CNS, when not found in the rest of the body at this time and largely absent from blood by 2 mo p.i., strongly suggests that the brain microenvironment favors the accumulation or survival of SIV-specific CD8+ T cells that enter the brain at very early time points after infection. To assess whether SIV persists in the brain despite the immune response to provide potential antigenic stimulation, two different regions of the brain were assessed for the presence of SIV RNA by the branched DNA assay. Low levels of SIV (averaging 34 copy equivalents of SIV per microgram of total brain RNA) could indeed be detected in both regions in all animals.

Viral escape

The disappearance of TatSL8-specific cells from the blood is due, at least in part, to viral escape (13, 14). To examine the role of viral escape, we molecularly cloned and sequenced the portion of the viral genome encoding the first exon of Tat from RNA purified from both plasma and the frontal lobe of the brain in the four chronically infected animals. A lower frequency of mutations within the TatSL8 epitope was found in the brain as compared with plasma (Table II). Each brain had a mixture of wild-type and mutant sequence, whereas the plasma of three of the animals, sequences encoding a wild-type TatSL8 epitope were rare or nonexistent. Interestingly, in two of the four animals, unique mutations were found in the brain, not being found contemporaneously in the plasma. These results, and the presence of high levels of TatSL8 CTL in the brain, suggest that the brain represents a unique compartment in the viral-host interaction during SIV infection, and that the maintenance of TatSL8 CTL in the brain may at least in part be due to persistence of the wild-type epitope. Although viral escape is likely one of the factors leading to the disappearance of TatSL8 in the blood, in our study, as well as those of others (13, 14), the wild-type epitope persists, albeit in the minority, in plasma virus of many animals; nevertheless, the TatSL8-reactive CTL disappear.


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  		Table II. Predicted amino acid sequencea

 
Early entry of CTL into the brain

Although we found SIV-specific CD8+ T cells in the CsF early following infection, the cerebrospinal fluid is a separate compartment from the brain, and may not accurately reflect the timing of entry of SIV-specific T cells into the brain. Because the peak of TatSL8-specific cells occurred in all animals at 28 days p.i., we inoculated the uninfected control MamuA*01 monkey with SIV, followed by sacrifice at this time point to assess the distribution of SIV-specific, tetramer-reactive CD8+ T cells in the brain relative to other organs. Strikingly, high levels of tetramer-reactive cells were present in the brain relative to other sites (of the CD8+ T cells in the brain, 27% reacted with the TatSL8 tetramer and 35% with the GagCM9 tetramer, whereas a maximum of 4% reactivity with either was found in the organs and fluids examined). Virus-specific cells thus enter in abundance in the brain early after infection and remain in the brain tissue even when, as exemplified by the TatSL8-reactive cells, such cells are no longer in the periphery.

Cytokines and the brain microenvironment

The brain microenvironment may also offer beneficial conditions for CTL accumulation and/or survival following their entrance in the brain. We therefore investigated the presence of cytokines (IL-2, IL-7, and IL-15) potentially involved in CTL survival (16) in the brains, spleen, deep cervical lymph nodes, and livers of the four chronically infected animals compared with six (except for liver, for which only five were available) uninfected controls (Fig. 4). SIV infection does not result in a change in IL-7 in any of the tissues examined. IL-2 is significantly increased in the liver (5.2-fold), but not detectable in either of the two brain regions examined. In contrast, IL-15 is significantly increased in both brain regions (2.6-fold in the hippocampus, and 2.2-fold in the frontal lobe), as well as the liver (1.9-fold), in SIV-infected animals.


Figure 4
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  		FIGURE 4. Cytokine levels in different organs. IL-2, IL-7, and IL-15 mRNA levels from the indicated regions of the brain, spleen, deep cervical lymph nodes, and liver were determined by quantitative reverse transcription real-time PCR, relative to the levels for control transcripts (18S, GAPDH, TBP), determined by the dCt method (relative units = 2dCt). Mean levels (±SEM) are shown. *, Significant difference (liver IL-2 p = 0.0434, liver IL-15 p = 0.0339, frontal lobe IL-15 p = 0.0055, hippocampus IL-15, p = 0.0156, two-tailed t tests).

 
To identify the cell type responsible for IL-15 production in the CNS, we performed immunohistochemical staining in the brain of the monkey sacrificed at 28 days p.i., whose levels of IL-15 mRNA were increased 6-fold (hippocampus) and 23-fold (frontal lobe) over control. Although immunoreactivity in neurons was relatively unchanged from that found in controls, greatly increased IL-15 expression was observed in astrocytes as well as blood vessels, including both the endothelium and perivascular macrophages (Fig. 5). The increase of this cytokine in the brain in the early stages of infection likely contributes to the early CNS enrichment of virus-specific cells and their eventual persistence.


Figure 5
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  		FIGURE 5. A–C, Immunohistochemical detection of CD8, IL-15, and GFAP at 28 days p.i. (animal 408). A, CD8+ cells can be found predominantly perivascularly; cells infiltrating the parenchyma are also present. B, IL-15 is found along the vasculature (endothelium and perivascular macrophages) and in cells of astrocytic morphology. C, GFAP labels activated astrocytes, a subset of which, as shown in B, express IL-15. D, Immunohistochemical detection of IL-15 in uninfected control brains (animal 382 shown) revealing lower level expression along the vasculature and in glial processes. In addition, IL-15 is expressed in neuronal subpopulations in control brains (as well as in the SIV-infected brains, without discernable changes in staining patterns). Insets, Lower left of each panel to reveal detail (at 4-fold increased magnification).

 
Role of IL-15 in sustaining CNS CTL

To test the ability of IL-15 to sustain brain-resident CD8+ T cells, we first cultured splenic and brain-derived cells from three SIV-infected but non-MamuA*01+ animals in the presence of different concentrations of IL-15. In both brain and spleen-derived cells, IL-15 induced an increase in numbers of CD8+ T cells. However brain-derived CD8+ T cells could expand in greater numbers, proliferating to significantly higher levels in response to increased levels of IL-15 (Fig. 6A).


Figure 6
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  		FIGURE 6. IL-15 and CTL proliferation. A, IL-15 supports growth of splenic as well as brain-derived CD8+ T cells. Values represent the fold increase of cell numbers (note log scale on y-axis) in 7-day cultures in the presence of the indicated concentration of cytokines relative to no added cytokine (mean ± SEM). Two-way repeated measures ANOVA analysis reveals a significant (p = 0.0085) effect of IL-15 concentration, as well as a greater response of cells from the brain than cells from the spleen (p = 0.0345). A Bonferroni post hoc test revealed that the 100 ng/ml concentration resulted in a significant difference between spleen and brain (*, p < 0.05). The ANOVA also revealed a significant interaction effect between organ and treatment (p = 0.0085), thus, the cell proliferation response is dependent upon dose, and is increased in the brain compared with spleen. B, Effect of IL-15 on CD95 expression. Splenic lymphoid cells were cultured in the presence of IL-15 (blue dots) or no added cytokine (red dots) and analyzed by flow cytometry after staining with reagents recognizing GagCM9-specific cells (y-axis) and CD95 (x-axis), revealing increased levels of CD95 following IL-15 treatment. Figure shows 50,000 events gated in the CD3+CD8+ compartment. C, Ki67 reactivity in spleen and brain, Gag and Tat tetramer+ cells in the four 6-mo-infected MamuA*01 animals (mean ± SEM). *, p = 0.04, two-tailed t test, for GagCM9-specific cells between brain and spleen; sufficient TatSL8-specific cells were not present in the spleen to allow analysis (N/A, not applicable). D, Representative FACS analysis revealing that a higher proportion of Gag (top) and Tat (bottom) tetramer+ CTL in the brain (left) express Ki67 expression than do those from spleen (right).

 
Although brain-derived cells proliferated to a much greater extent than did spleen cells in the presence of IL-15, we noted that on CD8+ T cells isolated from tissues, the expression of CD95/Fas was higher on brain-derived cells (average geometric mean fluorescent value ± SEM of 42.7 ± 2.6) than on those derived from blood (29.2 ± 4.2), spleen (33.9 ± 4.2), and deep cervical (24.1 ± 4.7) or inguinal (27.2 ± 5.0) lymph nodes, consistent with our previous findings (6). However, it has been demonstrated that IL-15 protects HIV-specific CTL from CD95/Fas-mediated apoptosis (17) and the higher level of CD95/Fas found on brain CTL may represent activation or further functional differentiation. In rhesus monkeys, it is well-established that CD95 expression is a marker for memory subsets of CD8+ T cells (18). To assess whether IL-15 may also play a role in the induction of CD95 on brain CTL, we cultured splenic CD8 cells from the four MamuA*01 chronically infected animals in the presence of IL-15 (100 ng/ml) or IL-2 (80 U/ml) for 7 days. The average mean fluorescence values within Gag tetramer+ cells for the expression of CD95 increased in response to IL-15 (72.0 ± 0.4), as compared with cells maintained with IL-2 (55.2 ± 4.3) or without added cytokines (40.1 ± 3.7) (Fig. 6B).

We next investigated whether there is evidence of CTL replication in the brain environment by measuring the expression of the proliferation nuclear marker Ki67 in brain-derived as well as splenic CD8 cells. Indeed, a large proportion of brain-derived cells were progressing in the cell cycle, as defined by Ki67 reactivity. This was present within the GagCM9 as well as TatSL8 tetramer-positive cell populations, and at a greater level than splenic CTL (Fig. 6, C and D). This suggests that the proximate source of brain CTL is not necessarily from migration from the blood, but they can self-renew in the brain environment, explaining persistency of clones long depleted from periphery.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
We have found that IL-15, known for its ability to support the maintenance of memory CD8+ T cells in the periphery, can play a similar role in the CNS. Early after SIV infection, when the CTL response to the TatSL8, which has a high functional avidity for SIV (14), quickly wanes in the blood, such cells enter the brain and persist. Interestingly, in influenza and dengue virus-infected mice, long-term persistence of virus-specific CTL in the brain has also been found (19, 20). Infection of mice with vesicular stomatitis virus also leads to the presence of memory CTL in the brain (21). When such mice were joined parabiotically with virus-naive mice, few cells appear capable of exiting the CNS and migrating to the brain of the conjoined mice, compared with CTL in other tissues (22). In a similar manner, TatSL8 CTL likely enter the CNS during the acute infection period and then are maintained in this environment.

Brain CTL are relatively uncharacterized in human disease. In the lentivirus-induced disease human T lymphotropic virus type I (HTLV-I)-associated myelopathy/tropical spastic paraparesis (for which HTLV-I-reactive CTL within the CNS take part in neuropathogenesis), IL-15 has been shown to play a key role in the maintenance of these CTL in PBMC (23). In both HTLV-I-associated myelopathy/tropical spastic paraparesis and SIV/HIV-related CNS sequela, IL-15 may support the CNS immune response. CTL within the brain microenvironment may control viral load, yet at the price of CNS dysfunction and potential development of inflammatory pathology. Potential neuropathogenic roles for CNS CTL have been postulated in other disorders such as multiple sclerosis (24), in which elevated IL-15 is found in the blood and CsF (25).

We propose that in the CNS, IL-15 supports the maintenance of virus-specific CTL, although other T cell growth factors such as IL-2 are not present. IL-15 has been found to support the growth of SIV-specific CTL in the absence of IL-2 in vitro (26). Indeed, when administered in vivo to rhesus monkeys, IL-15 enhances Ag-specific CTL (27, 28, 29). Furthermore, in mice it has been recently shown that following acute infection, the ability of CD8+ T cells to survive and become memory cells is dependent upon IL-15 (30). Therefore, the elevated levels of IL-15 in the CNS following SIV infection could be involved in maintaining local CTL, including clones that disappear from the rest of the body. The fact that brain-derived cells are more susceptible to IL-15 than cells from spleen may relate to a distinct subset of CD8+ T cells, as found in rodents, which is highly dependent upon IL-15 (31). Their potential enrichment in the brain vs the spleen would help explain the differential effect of IL-15 on brain CD8 T cells.

The uniqueness of the brain microenvironment is related to the particular cytokine environment in which IL-15 increases, but IL-2 is absent. This is different from the liver, where IL-15 increases but IL-2 increases to a much greater degree, or from lymphoid organs, where these cytokine levels remain unchanged. Other studies have demonstrated the importance of regional environment on the generation of CD8 cells that respond efficiently to viruses. For instance, in the intestine, mucosal immunization against simian/HIV in rhesus monkeys results in induction of high-avidity CTL in the intestinal lymphoid tissue, which are effective in delaying viral spread following intestinal challenge with virus (32, 33). Although the reason for this increased CTL avidity in intestinal mucosal immunization is not known, it is intriguing that administration of IL-15 with Ag leads to induction and survival of high-avidity CTL (34).

The control of CD8 memory cell numbers and their longevity has been found to be dependent on the balance between IL-15 and IL-2, being the expansion of these cells greatly enhanced in conditions of absence of IL-2 (35). The presence of elevated levels of IL-15 in the absence of IL-2 could thus support CD8 survival and replication uniquely in the brain, such as we find for the TatSL8 CTL. This may also help explain the persistence of activated CTL in the brain where replication-competent virus becomes undetectable, as in the model of intracerebral influenza injection in intranasally primed animals (19).

Basal levels of IL-15 are detected in various tissues, including the brain (36). Although its source is not well-characterized, neuronal cell lines can produce IL-15 (37). We indeed find immunoreactivity for IL-15 in neurons, although the up-regulation following SIV infection occurs in astrocytes. Astrocytic production of IL-15 can occur in response to viral infection and proinflammatory cytokines (38, 39) and appear to thus be able to control CD8 presence and accumulation in infected brains.

The early presence of antiviral CTLs in the brain likely contributes to a relatively low viral load in the brain, as suggested by our findings of a significant decline in viral RNA between 2 and 11 wk after SIV inoculation in rhesus monkeys (40) and the effects of early CD8 depletion, which lead to high brain viral RNA levels and early death (41, 42). Although we did not examine the possibility of latency, the role of the adaptive, as opposed to the innate, immune contribution to latency in the brain (43) is of interest for future studies.

In summary, we have found that CD8 cells specific to major immunodominant epitopes enter the brain very early after viral infection, and are maintained in the brain in the presence of increased IL-15, even when some clones are depleted from peripheral sites. Although at the outset, these CTL enter the brain to subdue expression of the virus and its expansion within the brain, their long-term persistence and activity can lead to untoward effects in the specialized CNS environment.


   Acknowledgments
 
We thank Dr. Bianca Mothé for helpful discussions, Nancy Delaney for administrative assistance and Cathie York-DeFalco and Ryan Ojakian for technical assistance. This is manuscript no. 18281 from The Scripps Research Institute.


   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 MH61224, MH73490, and MH62261. Back

2 Address correspondence and reprint requests to Dr. Maria Cecilia G. Marcondes, The Scripps Research Institute, 10550 North Torrey Pines Road, SP30-2030, La Jolla, CA 92037. E-mail address: cmarcond{at}scripps.edu Back

3 Abbreviations used in this paper: BAL, bronchioalveolar lavage; p.i., postinfection; GFAP, glial fibrillary acidic protein; Ct, cycle threshold; dCt, delta Ct; TBP, TATA box-binding protein; HTLV-I, human T lymphotropic virus type I. Back

Received for publication September 26, 2006. Accepted for publication February 6, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Hickey, W. F., B. L. Hsu, H. Kimura. 1991. T-lymphocyte entry into the central nervous system. J. Neurosci. Res. 28: 254-260. [Medline]
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