Alphavirus-Induced Encephalomyelitis: Antibody ... - Journal of Virology

JOURNAL OF VIROLOGY, Nov. 2011, p. 11490–11501 0022-538X/11/$12.00 doi:10.1128/JVI.05379-11 Copyright © 2011, American Society for Microbiology. All Rights Reserved.

Vol. 85, No. 21

Alphavirus-Induced Encephalomyelitis: Antibody-Secreting Cells and Viral Clearance from the Nervous System䌤 Talibah U. Metcalf and Diane E. Griffin* W. Harry Feinstone Department of Molecular Microbiology and Immunology, Johns Hopkins University Bloomberg School of Public Health, Baltimore, Maryland 21205 Received 11 June 2011/Accepted 16 August 2011

Sindbis virus (SINV) infection of the central nervous system (CNS) provides a model for understanding the role of the immune response in recovery from alphavirus infection of neurons. Virus clearance occurred in three phases: clearance of infectious virus (days 3 to 7), clearance of viral RNA (days 8 to 60), and maintenance of low levels of viral RNA (>day 60). The antiviral immune response was initiated in the cervical lymph nodes with rapid extrafollicular production of plasmablasts secreting IgM, followed by germinal center production of IgG-secreting and memory B cells. The earliest inflammatory cells to enter the brain were CD8ⴙ T cells, followed by CD4ⴙ T cells and CD19ⴙ B cells. During the clearance of infectious virus, effector lymphocytes in the CNS were primarily CD8ⴙ T cells and IgM antibody-secreting cells (ASCs). During the clearance of viral RNA, there were more CD4ⴙ than CD8ⴙ T cells, and B cells included IgG and IgA ASCs. At late times after infection, ASCs in the CNS were primarily CD19ⴙ CD38ⴙ CD138ⴚ Blimp-1ⴙ plasmablasts, with few fully differentiated CD38ⴚ CD138ⴙ Blimp-1ⴙ plasma cells. CD19ⴙ CD38ⴙ surface Igⴙ memory B cells were also present. The level of antibody to SINV increased in the brain over time, and the proportion of SINV-specific ASCs increased from 15% of total ASCs at day 14 to 90% at 4 to 6 months, suggesting specific retention in the CNS during viral RNA persistence. B cells in the CNS continued to differentiate, as evidenced by accumulation of IgA ASCs not present in peripheral lymphoid tissue and downregulation of major histocompatibility complex (MHC) class II expression on plasmablasts. However, there was no evidence of germinal center activity or IgG avidity maturation within the CNS. Alphaviruses of the family Togaviridae are an important cause of acute mosquito-borne viral encephalomyelitis in the Americas (7, 59). Neurons of the brain and spinal cord are the primary target cells, and recovery requires immune-mediated control of infection in these nonrenewable cells. Virus clearance from neurons poses unique challenges for the immune system. The restriction of the blood-brain barrier to immune effector entry into the central nervous system (CNS), reduced expression of major histocompatibility complex (MHC) classes I and II, and terminal differentiation of neurons make virus clearance more difficult (15). A noncytolytic process is needed to avoid irreversible neurologic damage, and the process must be effective to avoid chronic or progressive neurologic disease. Previous studies of immunodeficient mice infected with Sindbis virus (SINV), the prototype alphavirus, have shown that clearance of infectious virus from neurons within 7 to 8 days is mediated by gamma interferon (IFN-␥) produced by T cells and anti-E2 glycoprotein antibodies (Abs) produced by B cells (4, 23). Although infectious virus is cleared from the CNS to undetectable levels after infection, viral RNA encoding both structural and nonstructural viral proteins can be detected in the brains and spinal cords of SINV-infected BALB/c mice for at least a year after recovery (55, 22). In severe combined immu-

* Corresponding author. Mailing address: Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, 615 North Wolfe Street, Suite E5132, Baltimore, MD 21205. Phone: (410) 955-3459. Fax: (410) 955-0105. E-mail: dgriffin @jhsph.edu. 䌤 Published ahead of print on 24 August 2011.

nodeficiency (SCID) mice, production of infectious SINV resumes as levels of passively transferred Ab decrease, indicating that persistent RNA is capable of renewed replication (22). Persistence of viral RNA in the CNS suggests the need for long-term immune-mediated suppression of SINV reactivation after the acute phase of infection. Previous studies of BALB/c mice have shown that the acute inflammatory response to SINV infection includes the infiltration of T cells and B cells into the CNS (18, 40). Additional studies have shown that B-cell-deficient (␮MT) C57BL/6 mice are unable to clear infectious virus from cortical and hippocampal neurons and that initial successful SINV clearance from brain stem and spinal cord motor neurons is followed by virus reactivation after 18 to 22 days, demonstrating a critical role for Ab in recovery (4, 6). The presence of SINV-specific Ab-secreting cells (ASCs) in the brains of immunologically normal mice for at least a year after recovery from infection further suggests a role for intrathecal Ab production in the long-term suppression of virus reactivation (55). Together, these studies suggest that antiviral ASCs in the CNS are a critical aspect of the immune response to CNS virus infection. However, little is known about the phenotypes and changing functional characteristics of B cells in response to infection. We have used quantitative reverse transcription-PCR (qRT-PCR) to measure changes in the levels of viral RNA after the clearance of infectious virus and have shown a 6- to 8-week period of decreasing RNA levels, followed by many months of stable low levels of viral RNA. Simultaneously, we have documented the entry and retention of T cells and B cells and have characterized the phenotypes and functions of ASCs in the CNS. Clearance of infectious virus occurs

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prior to the production of antiviral IgG and correlates with the infiltration of CD8⫹ T cells and the presence of ASCs producing antiviral IgM. Subsequent clearance of viral RNA and suppression of renewed virus replication correlates with the infiltration of CD4⫹ T cells and the presence of ASCs producing antiviral IgG. MATERIALS AND METHODS Mice and virus infection. Female 4- to 6-week-old C57BL/6J mice were obtained from the Jackson Laboratory (Bar Harbor, ME). Male and female heterozygous Blimp-1GFP/⫹ transgenic C57BL/6J mice, expressing green fluorescent protein (GFP) under the control of Blimp-1 regulatory elements (19), were bred locally. Mice were infected at the age of 4 to 6 weeks by intracerebral inoculation with 1,000 PFU of TE, a recombinant strain of SINV (26). Control mice were inoculated with phosphate-buffered saline (PBS). All procedures were performed in compliance with protocols approved by the Johns Hopkins University Animal Care and Use Committee. Infectious virus and virus RNA assays. Tissue homogenates (20% [wt/vol]) were prepared from the whole spinal cord (⬃0.1 g of tissue) and the left hemisphere of the brain (⬃0.2 g of tissue) of 3 to 4 mice. Tissue was placed in a DNA-lysing matrix A tube (MP Biomedicals) and was dissociated with 1 ml of 1% fetal bovine serum (FBS)-Dulbecco’s modified Eagle’s medium (DMEM) using a FastPrep-24 homogenizer (MP Biomedicals) at setting 4 for 40 s. Tubes were centrifuged for 10 min at 13,000 rpm (4°C), and the clarified supernatant fluid was stored at ⫺80°C. Brain and spinal cord homogenates were serially diluted 10-fold and were assayed for infectious virus by plaque formation on BHK cells. RNA was isolated from the whole spinal cord and half of a brain hemisphere (⬃0.1 g of tissue) of 3 to 4 mice by using the Qiagen RNeasy Lipid Tissue Mini kit. SINV cDNA was synthesized using the SuperScript first-strand synthesis kit (Invitrogen) and primers for the minus (5⬘-CAC GGC AAT GTG TTT GCT-3⬘-nt 8456) and plus(5⬘-AGC ATT GGC CG ACCT AAC GCA GCA C-3⬘-nt 9899) strands of the SINV genome. Quantitative real-time PCR was performed using 2.5 ␮l of cDNA, TaqMan Universal PCR Master Mix, and TaqMan probe nt 8760-5⬘–6-carboxyfluorescein (FAM)-CGC ATA CAG ACT TCC GCC CAG T–6-carboxytetramethylrhodamine (TAMRA)-3⬘-8781 (Applied Biosystems). The following primers amplified the E2 structural region gene: forward, nt 8732-5⬘-TGG GAC GAA GCG GAC GAT AA-3⬘-nt 8752; reverse, nt 8805-5⬘-CTG CTC CGC TTT GGT CGT AT-3⬘-nt 8786. Reactions were performed in triplicate on a 7500 Fast real-time PCR system and were analyzed by absolute quantitation using Sequence Detector software, version 1.4 (Applied Biosystems). PCR conditions were as follows: 2 min at 50°C and 10 min at 95°C, followed by 40 cycles, where 1 cycle consisted of 15 s at 95°C and 1 min at 60°C. Virus RNA copies were determined based on a standard curve, used for threshold cycle (CT) values, and run in parallel using serial 10-fold dilutions of a pGEM-3Z plasmid containing the E2 coding region. The absence of a template was used as a negative control. Isolation of mononuclear cells. Brains (pooled from 6 mice) were homogenized using the Neural Tissue Dissociation kit with trypsin along with gentleMACS C Tubes and a gentleMACS dissociator (Miltenyi Biotec). The dissociated tissue was incubated at 37°C with DNase and collagenase, followed by filtration through a 70-␮m-pore-size cell strainer washed with cold 0.5% bovine serum albumin (BSA)–Hanks balanced salt solution without Ca2⫹ and Mg2⫹ (HBSS). Cells were pelleted at 1,200 rpm for 10 min and were resuspended in cold 0.9 M sucrose in HBSS with Ca2⫹ and Mg2⫹ to separate the cells from myelin. After centrifugation at 850 ⫻ g for 10 min with slow braking, the cell pellet was washed in cold HBSS with Ca2⫹ and Mg2⫹. Cervical lymph nodes (CLNs) (pooled from 6 mice) and spleens (pooled from 2 mice) were homogenized in cold PBS–2 mM EDTA–0.5% BSA (PEB) using gentleMACS C Tubes and a gentleMACS dissociator. Bone marrow (pooled from 2 mice) was collected from femurs and tibiae by flushing the shaft with cold PEB. Peripheral tissue cell suspensions were filtered through a 70-␮m-pore-size strainer and were pelleted at 1,200 rpm for 10 min. Cell pellets were resuspended in 2 to 3 ml of ammonium chloride (Sigma) for 3 min to lyse contaminating red blood cells. The lysis reaction was quenched by the addition of either BSA–HBSS (brain) or PEB (peripheral tissue), and cells were pelleted, resuspended in PEB, and counted. Flow cytometry. All staining was performed in a 96-well round bottom plate with 106 cells per well. Dead cells were distinguished using the Live/Dead Fixable Dead Cell Stain kit (Invitrogen). Fc␥ receptors were blocked using anti-mouse CD16/CD32 (Miltenyi Biotec) diluted in PEB. Cells were stained with antibodies to surface markers for 30 min on ice, followed by intracellular staining using the BD Cytofix/Cytoperm kit. Abs were diluted 1:100 in PEB for surface staining and

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in BD Perm/Wash for intracellular staining. The monoclonal Abs (BD Biosciences) used were against CD19 (clone 1D3), CD3 (clone 17A2), CD4 (clone RM4-5), CD8 (clone 53-6.7), CD38 (clone 90/CD38), CD138 (clone 281-2), IgM (clone R6-60.2), IgD (clone 11-26c.2a), IgG2a/IgG2b (clone R2-40), IgA (clone C10-3), I-A/I-E (clone M5/114.15.2), and T- and B-cell activation antigen (clone GL7). Cells were analyzed on a BD FACSCanto II system with FACSDiva software, version 6. A total of 100,000 events were collected, and statistical analysis was performed with FlowJo software, version 9. Detection of Ab-secreting cells. Total and SINV-specific ASCs in the brain and peripheral tissue were quantified by an enzyme-linked immunosorbent (ELISPOT) assay. Ninety-six-well Multiscreen cellulose plates (Millipore) were coated with either goat anti-mouse IgM plus IgG plus IgA (H⫹L) (Southern Biotech) or a SINV-infected BHK cell lysate diluted in 50 mM NaHCO3 (pH 9.6) to detect total ASCs or SINV-specific ASCs. NaHCO3 buffer and an uninfected BHK cell lysate were used as negative controls. Plates were blocked with 10% FBS–RPMI-1640 medium for 2 h at 37°C. Mononuclear cells, isolated as described above, except that buffers contained 1% FBS, were pooled from 6 mice for the CLNs and brain and from 2 mice for the spleen and bone marrow. Cells were resuspended in 10% FBS–RPMI-1640 medium, plated in triplicate (5 ⫻ 105 cells/well for the brain; 106 cells/well for peripheral tissues), and incubated for 5 h at 37°C under 5% CO2. Plates were washed with PBS–1% FBS–0.1% Tween 20 (wash buffer) to remove cells and were incubated overnight at 4°C with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG1, IgG2a, IgG2b, IgG3, IgM, or IgA (Southern Biotech) diluted 1:2,000 in wash buffer. Plates were washed, and spots were developed with diaminobenzidine (DAB) substrate (Invitrogen) in the dark. Spots were counted using an automated ELISPOT reader (CTL ImmunoSpot analyzer) and were reported as spot-forming cells/106 cells. IgG1, IgG2a, IgG2b, and IgG3 spot counts were combined to generate total and SINV-specific IgG ASC numbers. The percentages of IgM, IgG, and IgA ASCs that were SINV specific were calculated by using the total spots counted for each isotype as the denominator. The percentages of SINV-specific ASCs secreting each IgG subclass were calculated by using total SINV-specific ASCs as the denominator. Negative controls for each tissue had ⬍5 spot-forming cells/106 cells for all isotypes. Ab assays. SINV-specific IgM, IgG, and IgA and Ab avidity were assessed by an enzyme immunoassay. Ninety-six-well Maxisorp plates (Nalgene Nunc) were coated overnight at 4°C with a lysate of SINV-infected BHK cells diluted in 50 mM NaHCO3 (pH 9.6). Brain homogenates were prepared as for plaque assays, and sera were pooled from 4 to 6 mice. Using blocking buffer (10% FBS, 0.05% Tween 20 in PBS), homogenates were diluted 1:2, and sera were diluted 1:100 and were added to coated wells. Bound antibody was detected with HRP-conjugated goat anti-mouse IgM, IgG, and IgA (Southern Biotech) using 3,3,5,5-tetramethylbenzidine (TMB) as the substrate (Sigma). Absorbance was read at 450 nm. Optical density (OD) measurements from day zero were subtracted, and the resulting values were plotted as the mean OD ⫾ the standard error of the mean (SEM). An anti-SINV E2 positive control was included in each assay. To determine Ab avidity, plates were additionally incubated with increasing concentrations of ammonium thiocyanate (NH4SCN) (0 to 3.5 M) at room temperature for 15 min. The avidity index for each sample was defined as the concentration of NH4SCN required to reduce Ab binding by 50% (37, 27). Samples with OD values of ⬍0.3 without NH4SCN were not analyzed for avidity.

RESULTS Clearance of SINV from the CNS. To determine the time course of virus replication and clearance from the CNS, infectious virus and viral RNA in the brains and spinal cords of SINV-infected B6 mice were quantified (Fig. 1A). Both infectious virus and SINV RNA levels were maximal at day 3. Infectious virus was cleared from both the brain and the spinal cord to undetectable levels by day 7. Viral RNA levels declined steadily over 2 months and then persisted at a low level for at least 6 months. Thus, three phases of immune control of virus replication in the CNS were apparent: clearance of infectious virus (day 3 to 7), clearance of viral RNA (day 8 to 60), and maintenance of low levels of viral RNA (⬎day 60).

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FIG. 1. Virus clearance and T-cell and B-cell infiltration into the CNS. (A) Infectious virus (PFU/gram of tissue) and viral RNA (copies/2.5 ␮g cDNA) in the brain and spinal cord. Horizontal line represents the limit of detection of RNA. Data are plotted as the geometric means for 3 to 4 mice ⫾ SEM. (B to D) Mononuclear cells isolated from pooled brains of infected mice (n, 6 to 8) were analyzed for CD19, CD3, CD4, and CD8 expression by flow cytometry. (B) Flow cytometry gating scheme used to detect B and T cells within the gated lymphocyte population and CD4 and CD8 T cells within the gated CD3⫹ population. (C) Numbers of CD19⫹ B cells and CD3⫹ T cells gated within 100,000 events. (D) Frequencies (expressed as percentages) of CD4⫹ T cells and CD8⫹ T cells within the CD3⫹ T cell population. Each point in panels C and D represents the mean ⫾ SEM for 3 to 4 independent experiments.

Lymphocyte recruitment into the CNS. Flow cytometry was used to monitor lymphocyte entry and retention in the brains of SINV-infected mice. CD3⫹ T cells infiltrated the brain earlier and in larger numbers than CD19⫹ B cells (Fig. 1B and C). CD3⫹ T cells were detected by day 3, and their numbers increased rapidly to a maximum at day 10. B-cell numbers were also maximal at day 10 but increased more slowly. The time courses of the entry of CD4⫹ and CD8⫹ T cells differed (Fig. 1D). CD8⫹ T-cell numbers were maximal at day 5 and then declined, while CD4⫹ T cells appeared later, were maximal at day 10, and remained more numerous than CD8⫹ T cells for

the rest of the study period. Thus, a shift from CD8⫹ to CD4⫹ T cells occurred after the clearance of infectious virus, and the increase in CD4⫹ T cell levels coincided with an increase in B-cell levels. Production of SINV-specific Abs in peripheral lymphoid tissue. ASCs are generated in secondary lymphoid tissue in two phases: an early extrafollicular response that produces primarily low-affinity IgM and a later, T-cell-dependent germinal center (GC) follicular response that produces high-affinity IgG and IgA (2). To assess when and where SINV-specific ASCs are first produced, draining CLNs and spleens were analyzed

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FIG. 2. Peripheral IgM, IgG, and IgA responses. (A through F) The numbers of total (A, C, and E) and SINV-specific (B, D, and F) ASCs secreting IgM (A and B), IgG (C and D), and IgA (E and F) in the cervical lymph node (CLN) (pooled from 6 to 8 mice), spleen (pooled from 2 mice), and bone marrow (BM) (pooled from 2 mice) were measured by ELISPOT assays. Data are means ⫾ SEM for 2 to 3 independent experiments (except for measurement of SINV-specific ASCs on day 5, when 1 experiment was conducted). (G) Measurements of the OD at 450 nm of SINV-specific Abs in serum (1:100). Each point represents data from sera pooled from 4 to 6 mice.

by an ELISPOT assay (Fig. 2). In the CLNs there was an increase in the number of IgM-secreting ASCs that peaked at 5 to 7 days (Fig. 2A) and coincided with the appearance of SINV-specific IgM ASCs during the extrafollicular response

(Fig. 2B). Total and SINV-specific IgG ASCs developed in the CLNs by 5 days after infection and were maximal at day 10, coinciding with the germinal center response (Fig. 2C and D). Few total or SINV-specific IgA ASCs were detected in the

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FIG. 3. CNS IgM, IgG, and IgA responses. (A and B) The numbers of total (A) and SINV-specific (B) IgM, IgG, and IgA ASCs in the brain (pooled from 6 to 8 mice) were measured by an ELISPOT assay. (C) Percentages of total IgM, IgA, and IgG ASCs that were specific against SINV. (D) Percentages of SINV-specific IgG subclass ASCs within the total number of SINV-specific IgG ASCs. Data are means ⫾ SEM from 2 to 3 independent experiments (except for day 7, on which 1 experiment was conducted). (E) Measurements of the OD at 450 nm of SINV-specific Abs in brain homogenates diluted 1:2. (F) Avidity indices for SINV-specific IgM, IgG, and IgA Abs in brain homogenates diluted 1:10. Each point is the mean ⫾ SEM for 3 mice.

CLNs during this time (Fig. 2E and F). SINV-specific IgM and IgG ASCs were not present at high frequencies in the spleen during this period, indicating that the primary site for induction of the adaptive immune response to SINV CNS infection is in the CLNs. After resolution of the acute phase of infection, the bone marrow is often a major site for the long-term maintenance of ASCs, which are the main contributors of serum Abs (28, 42). SINV-specific IgG and IgA ASC levels increased in the bone marrow beginning 1 month after infection (Fig. 2D and F). SINV-specific IgM and IgG Abs were detected in the serum by day 10 to 14 (Fig. 2G). SINV-specific IgG Ab was the main Ab detected after 1 month. SINV-specific IgA Ab remained at low levels in the serum, paralleling the levels of SINV-specific IgA ASCs in the CLNs, spleen, and bone marrow. Production of SINV-specific Abs in the brain. ASCs exiting the CLNs can enter the blood and migrate to sites of inflammation. Total and SINV-specific IgM ASCs were detected in the brain 7 days after infection (Fig. 3A and B). SINV-specific IgM ASCs were maximal at day 10 and were maintained at a high frequency in the brain for 2 months before beginning to decline. SINV-specific IgG ASCs were first detected at day 10, increased steadily, and became equivalent to SINV-specific IgM ASCs by 4 months after infection (Fig. 3B). IgG2a and IgG2b were the most abundant IgG subclasses for SINV-specific ASCs in the brain (Fig. 3D), CLNs, and spleen (data not shown). Although SINV-specific IgA ASCs were infrequent in the CLNs, they were readily detected within the CNS (Fig. 3B). IgA-producing cells peaked at days 10 to 14, and their numbers were maintained for at least 6 months after infection. Total numbers of ASCs decreased along with a general decrease in

inflammation after the clearance of infectious virus (Fig. 3A), but the numbers of SINV-specific ASCs increased steadily (Fig. 3B). Thus, the percentage of ASCs that were SINV specific increased from 15% at day 14 to 90% at 2 to 6 months (Fig. 3C). These data suggest that SINV-specific ASCs were retained in the brain, while nonspecific ASCs died and/or left the brain. The amounts of SINV-specific Ab being produced in the brain over the course of the study were measured by an enzyme immunoassay (EIA) using brain homogenates (Fig. 3E). SINV-specific IgM Ab peaked at 10 to 14 days and was undetectable after 1 month. SINV-specific IgA Ab reached maximum levels at 1 month and remained detectable for at least 6 months. SINV-specific IgG Ab was first detected at day 14 and increased steadily, reaching maximum levels at 4 months. These data indicate that the SINV-specific IgM ASCs remaining in the brain after recovery produce low levels of Ab in comparison to IgG and IgA ASCs. To determine whether avidity maturation of the Abs being produced in the brain was occurring, avidity indices were determined for the Abs in brain homogenates (Fig. 3F). SINVspecific IgM produced early was of low avidity, while IgA and IgG, which were present 1 month after infection, showed substantially higher avidity, but this did not increase with time. These data suggest that SINV-specific ASCs do not continue the germinal center process of somatic hypermutation and selection after entering the CNS. Changes in B-cell expression of sIgD and sIgM. To further characterize the B-cell response to SINV infection, CD19⫹ B cells were analyzed by flow cytometry for expression of differentiation markers. Naïve B cells express both surface IgM

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FIG. 4. Generation and recruitment of activated B cells. Mononuclear cells isolated from pooled CLNs and brains of infected mice (n ⫽ 6) were analyzed for CD19, sIgM, and sIgD expression by flow cytometry. (A) Flow cytometry gating scheme for detecting sIgM⫹ and sIgD⫹ B cells within the CD19⫹ population. (B and C) Numbers of CD19⫹ cells expressing sIgM and sIgD in the CLN (B) and brain (C). Graphs show the number of B cells gated within 100,000 events, and each point represents the mean ⫾ SEM for 3 to 4 independent experiments.

(sIgM) and sIgD. After antigen stimulation, sIgD is downregulated, and only sIgM continues to be expressed. After germinal center differentiation and class switch recombination for IgG and IgA production, sIgM is no longer expressed (21). Therefore, staining for sIgM and sIgD identifies naïve and activated unswitched and switched B-cell populations (Fig. 4A). In the CLNs, the numbers of naïve (sIgD⫹) B cells were abundant and relatively stable throughout the analysis period (Fig. 4B). Activated switched (sIgM⫺ sIgD⫺) B cells showed the greatest increase in numbers, were maximal by day 14, and returned to baseline by 2 months after infection, consistent with the CLN as the site for induction of the adaptive immune response to SINV. In the brain, both naïve and activated B cells were detected by day 7 and peaked at day 10 after infection (Fig. 4C). However, activated switched (sIgM⫺ sIgD⫺) B cells were the main subset that persisted above baseline for at least 6 months. Activated sIgM⫹ sIgD⫺ unswitched B cells and naïve B cells also infiltrated the CNS but were not maintained, either because they died, left, or were differentiated further. Thus,

the majority of the B cells residing in the SINV-infected brain have undergone class switch recombination to become ASCs or memory B cells. Few terminally differentiated ASCs in the CNS. To determine the maturation state of activated B cells, the expression of CD38 and CD138 was analyzed (Fig. 5). Murine CD38 is expressed on naïve and activated B cells (including plasmablasts [PBs] and memory B cells) and is downregulated on germinal center B cells and mature plasma cells (PCs) (25, 39, 35). During ASC differentiation, CD138 is upregulated by Blimp-1, committing the cells to become PCs, while memory cells do not express Blimp-1 (1, 43). Terminally differentiated PCs usually become CD19⫺, while earlier proliferating ASCs remain CD19⫹ (30). Therefore, to determine the state of ASC differentiation, both CD19⫹ and CD19⫺ CD3⫺ cells were analyzed for CD38 and CD138 expression (Fig. 5). In the CLNs, the number of CD19⫹/⫺ CD138⫹ mature PCs peaked between days 5 and 7 after infection and returned to baseline by 1 month (Fig. 5A and B). In the brain,

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FIG. 5. Plasmablasts and plasma cells in the CLNs and brain after infection. Mononuclear cells isolated from pooled CLNs and brains of infected C57BL/6J mice (n ⫽ 6) and Blimp-1GFP/⫹ reporter mice (n ⫽ 2) were analyzed for CD19, CD38, CD138, and GFP expression by flow cytometry at various times after infection. (E) Flow cytometry gating scheme for detecting CD38 and CD138 subpopulations within the CD19⫹ population. (A and B) Numbers of CD19⫹ CD38⫺ CD138⫹ (A) and CD19⫺ CD38⫺ CD138⫹ (B) ASCs in the CLNs. (C, D, and F) Numbers of CD19⫹ CD38⫺ CD138⫹ (C) and CD19⫺ CD38⫺ CD138⫹ (D) plasma cells and CD19⫹ CD38⫹ CD138⫺ plasmablasts (F) in the brain. Graphs show the numbers of B cells gated within 100,000 events, and each point represents the mean ⫾ SEM for 3 to 4 independent experiments. (G and H) Dot plots represent CD19⫹ CD138⫹ GFP⫹ plasma cells and CD19⫹ CD38⫹ GFP⫹ plasmablasts within the CLNs (G) and brain (H) at 10 days and 2 months after infection.

CD19⫹/⫺ CD38⫺ CD138⫹ mature PCs were infrequent and changed little over the course of infection (Fig. 5C and D). In contrast, there was a marked increase in the numbers of more immature CD19⫹ CD38⫹ CD138⫺ B cells infiltrating the brain in response to SINV infection, with maximal numbers at day 10 (Fig. 5F). To further characterize ASCs in the brain, B cells were analyzed for the expression of CD93, which is upregulated on terminally differentiated PCs.

CD19⫹/⫺ CD138⫹ CD93⫹ cells were identified in the CLNs, but very few were detected in the brain (data not shown). Also, we infected Blimp-1⫹/GFP reporter mice (19) with SINV and assessed GFP expression by B cells (Fig. 5G and H). In the CLNs, GFP⫹ B cells expressed both CD38 and CD138, indicating the presence of both PBs and PCs (Fig. 5G). However, in the brain, most GFP⫹ B cells expressed CD38, and only a few expressed CD138 (Fig. 5H). Thus,

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FIG. 6. Generation and recruitment of cIg⫹ ASCs and sIg⫹ memory B cells. Mononuclear cells isolated from pooled CLNs and brains of infected mice (n ⫽ 6) were analyzed for expression of CD19 and either cytoplasmic (c) or surface (s) IgG2a/IgG2b and IgA by flow cytometry. (A and D) Flow cytometry gating schemes for detecting sIgG⫹ and cIgG⫹ cells in the CLN (A) and brain (D) CD19⫹ populations. (B and E) Numbers of CD19⫹ sIgG⫹ memory (BMem) and CD19⫹ cIgG⫹ (ASCs) B cells in the CLNs (B) and brain (E). Graphs show the number of B cells gated within 100,000 events, and each point represents the mean ⫾ SEM for 2 independent experiments. (C and F) Numbers of CD19⫹ sIgA⫹ memory (BMem) and CD19⫹ cIgA⫹ (ASCs) B cells in the CLNs (C) and brain (F). Each point represents the mean ⫾ SEM for 2 independent experiments (except for days 0 and 180, on which 1 experiment was conducted).

most of the ASCs that are maintained in the brain are PBs and not terminally differentiated PCs. ASCs and memory B cells traffic to the CNS. CD19⫹ CD38⫹ GFP⫺ B cells may be memory B cells involved in replenishing the PB population. During differentiation, ASCs downregulate surface immunoglobulin and increase the production of intracellular immunoglobulin (5, 29). Memory B cells have small amounts of intracellular immunoglobulin until expansion and differentiation into ASCs (31). Therefore, to determine the proportion of ASCs and memory B cells, B cells were assessed by flow cytometry for either intracellular IgG2a/IgG2b (cIgG2a/cIgG2b) or sIgG2a/sIgG2b (IgG) and cIgA or sIgA

expression (Fig. 6A and D), with the hypothesis that the majority of cIg⫹ cells are ASCs while the majority of sIg⫹ cells are memory cells. In the CLNs, CD19⫹ sIgG⫹ memory B cells and cIgG⫹ ASCs peaked 10 to 14 days after infection (Fig. 6B), while CD19⫹ sIgA⫹ memory B cells and cIgA⫹ ASCs appeared unchanged during the acute phase of the infection (Fig. 6C). More CD19⫹ cIgG⫹ ASCs infiltrated the brain than sIgG⫹ memory B cells, but both subsets reached maximum levels 10 to 28 days after infection (Fig. 6E). During this time which coincided with the detection of CD38⫹ CD138⫺ B cells in the brain (Fig. 5F), 80% of CD19⫹ cIgG⫹ and sIgG⫹ B cells in the brain were CD38⫹ (data not shown). CD19⫹ cIgA⫹

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FIG. 7. MHC class II expression on cIgG⫹ ASCs and activated germinal center B cells. Mononuclear cells isolated from pooled CLNs (A to C) and brains (D to E) of infected mice were analyzed for cytoplasmic IgG2a/IgG2b and MHC class II (n, 4) or CD38 and GL7 (n, 6 to 8) by flow cytometry. (A and D) Flow cytometry gating schemes for detecting MHC II ASCs (A) and GL7 cells (D) in the CLN and brain CD19⫹ populations. (B and C) Numbers of CD19⫹ cIgG⫹ MHC II⫹ and CD19⫹ cIgG⫹ MHC II⫺ ASCs in the CLNs (B) and brain (C). PBS-inoculated mice (n ⫽ 4) were used as a negative control at day 10. Graphs show the number of B cells gated within 100,000 events, and each point represents the mean ⫾ SEM for duplicate staining from one experiment. (E and F) Numbers of CD19⫹ CD38⫺ GL7⫹/High and CD19⫹ CD38⫹ GL7⫹/High B cells in the CLNs (E) and brain (F). Each point represents the mean ⫾ SEM for 2 independent experiments.

ASCs were more abundant than sIgA⫹ memory B cells, which reached maximum levels 10 to 28 days after infection (Fig. 6F), like cIgG⫹ ASCs. Thus, both PBs and memory B cells enter and are retained in the CNS in response to SINV infection. Further differentiation of cIgGⴙ ASCs in the absence of local germinal center activity in the CNS. To further characterize the differentiation of cIgG⫹ ASCs, we analyzed MHC class II expression, which is downregulated on differentiating ASCs (42) (Fig. 7A). In the CLNs, a majority of cIgG⫹ ASCs expressed MHC class II, with little evidence of downregulation over time (Fig. 7B). In the brain, a majority of cIgG⫹ ASCs also expressed MHC class II at day 10 (Fig. 7C). However, the frequency of MHC II⫺ cIgG⫹ ASCs increased at day 32, and

by 6 months, similar numbers of cIgG⫹ ASCs were MHC II⫹ and MHC II⫺. These data suggest that in situ differentiation of cIgG⫹ ASCs occurs in the CNS. ASC differentiation could be occurring in ectopic lymphoid follicles in the brain serving for local generation of B-cell responses, as is the case in the autoimmune demyelinating disease multiple sclerosis (10, 41). To determine if functional activities similar to those in germinal centers occur in the brain following SINV infection, we analyzed the expression of CD38 (expected to be downregulated) and GL7 (expected to be upregulated) (9, 33, 35) (Fig. 7D). In the CLNs, CD38⫹/⫺ GL7⫹/High B cells peaked between days 10 and 14 after infection (Fig. 7E). GL7⫹/High B cells were not present in the spleen (data not shown), consistent with the

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notion that the CLN is the site for induction of the adaptive immune response to SINV. CD38⫹ GL7⫹/High B cells reached maximum levels in the brain at day 10 after infection and returned to baseline by 2 months (Fig. 7F). This coincides with the peak of CD19⫹ CD38⫹ CD138⫺ B cells (Fig. 5F), suggesting that germinal-center-derived B cells from the CLN are infiltrating the brain and are then undergoing further differentiation in the absence of local germinal center activity. DISCUSSION These studies have shown that in vivo clearance of SINV from neurons occurs in three phases: rapid clearance of infectious virus, followed by gradual clearance of viral RNA and then maintenance of low levels of viral RNA. Correlation of the immune response with this clearance process showed that the adaptive immune response is initiated in the draining CLNs and that CD8⫹ T cells enter the brain first, followed by CD4⫹ T cells and CD19⫹ B cells. During the clearance of infectious virus, the predominant effector cells in the brain were CD8⫹ T cells and extrafollicular PBs secreting SINVspecific IgM. During the clearance of viral RNA and the prevention of reactivation, the most abundant inflammatory cells were CD4⫹ T cells and SINV-specific IgG⫹ PBs and memory B cells. B-cell maturation continued in the CNS with a downregulation of MHC class II expression and a class switch recombination to IgA ASCs, but there was no evidence of local germinal center activity or avidity maturation. Entry of inflammatory cells into the CNS from the periphery is essential for the clearance of infectious virus (14, 34), and previous studies have shown that noncytolytic effectors of virus clearance include IFN-␥ and antiviral IgG (4, 23). CD8⫹ T cells, a likely source of locally produced IFN-␥, peaked at day 5 after infection, when clearance is initiated, and are important for the clearance of other CNS virus infections (44, 45, 49, 57). However, studies of ␮MT mice have shown that T cells alone cannot clear infectious SINV from many regions of the brain and that Ab is also required (4, 6). Our previous studies using passive transfer of monoclonal Abs to persistently infected SCID mice and in vitro studies of Ab-mediated suppression of virus production by differentiated neurons have shown that IgG Ab to the SINV E2 glycoprotein is effective in clearing infectious virus (23, 56). However, in the current study, infectious virus was cleared by day 8, even though infiltrating ASCs did not produce antiviral IgG until day 10 (54). The appearance of virus-specific IgG and IgA ASCs after the clearance of infectious virus was also observed in the CNS of mice infected with mouse hepatitis virus (52). Extrafollicularly derived SINV-specific IgM ASCs were the first to arrive in the brain and were detectable by day 5. The presence of these cells early after infection supports previous studies showing that extrafollicular ASCs can leave the lymph nodes to home to other organs and suggests an important role for IgM in clearance (13, 58). Previous data from T-cell-deficient athymic nu/nu mice suggest that locally produced IgM is sufficient for virus clearance. These mice lack mature T cells and produce virus-specific IgM and little IgG but are able to clear virus from the CNS with normal kinetics (16, 54). Antiviral IgM ASCs also play a role in the early control of West Nile virus dissemination to the CNS from the periph-

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ery (12). These data suggest that local IgM-producing ASCs, rather than IgG-producing ASCs, cooperate with IFN-␥producing CD8⫹ T cells for the clearance of infectious virus from the CNS. However, clearance of infectious virus does not mean clearance of viral RNA from terminally differentiated neurons. Previous studies detected persistent viral RNA by RT-PCR in the CNS of BALB/c mice infected with SINV strain AR339, but the amounts of RNA were not quantified to determine whether levels changed over time (55). In situ hybridization on brains from SCID mice treated with passive transfer of SINV Ab showed a gradual decrease in viral RNA levels, but these mice lacked T cells (23). The current quantitative studies showed that viral RNA remained at high levels for many days after clearance of infectious virus and was only gradually cleared from the brain and spinal cord over the next several weeks. Previous studies have shown that the period between 2 and 4 weeks after infection is frequently a time for virus reactivation (6) and suggest that decreasing the levels of viral RNA during this period is critical for recovery. The clearance of virus RNA coincided with the increase in SINV-specific IgG and IgA ASCs and Ab levels in the CNS after day 10. SINV-specific IgM ASCs in the brain were still numerous after 1 month. However, IgM Ab levels declined after day 14, suggesting that these late IgM ASCs were secreting only small amounts of Ab. In addition, the numbers of CD8⫹ T cells in the CNS began contracting after infectious virus was cleared (day 7), while CD4⫹ T cells were still increasing. Mice deficient in IFN-␥ or in the response to IFN-␥ often display transient reactivation of virus production 12 to 22 days after infection (4), suggesting an important role for IFN-␥ during this phase of clearance. We hypothesize that type 1 CD4⫹ T cells are essential for continued local production of IFN-␥ and potentially for providing local help to maintain both CD8⫹ T-cell function (3, 50, 60) and Ab production by B cells (36). In lymphoid tissue, isotype switching is facilitated by interaction between B cells and CD4⫹ T cells (47, 11), and in the bone marrow, the persistence of long-lived PCs is dependent on CD4⫹ T cells (51). The influence of type 1 CD4⫹ T cells during this period is further suggested by the preferential production of IgG2a and IgG2b rather than IgG1 (32, 46). Therefore, CD4⫹ T-cell persistence after the clearance of infectious virus could also be involved in the differentiation and retention of antiviral ASCs in the CNS. The individual roles and mechanisms of IFN-␥, IgG, and IgA in the clearance of viral RNA from neurons will require further experimentation. Phenotyping of ASCs using flow cytometry revealed that terminally differentiated PCs made up only a small percentage of B cells in the CNS. PBs were the main ASC population infiltrating and being maintained in the brain, along with memory B cells. The use of reporter mice to analyze the expression of Blimp-1, a key regulator of PC differentiation (1, 24), supported this conclusion. CD19⫹ CD38⫹ B cells expressed Blimp-1, indicating that these cells were committed to the PC lineage, but the lack of CD138 expression indicates that the ASCs infiltrating the brain remained immature. A large proportion of infiltrating B cells undergo apoptosis and/or leave the brain after the clearance of infectious virus, as evidenced by the sharp decline in the numbers of total ASCs and CD19⫹ B cells after day 14. However, the numbers of

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SINV-specific ASCs in the brain continued to increase up to 2 months after infection, when cells are no longer being produced in the CLNs and the blood-brain barrier function is normal, suggesting retention rather than continued recruitment. The continual increase in the numbers of SINV-specific ASCs could be due to in situ differentiation in the CNS. This possibility is supported by the steady increase in frequency of CD19⫹ cIgA⫹ B cells in the CNS and the absence of SINVspecific IgA ASCs in the CLNs and of IgA Ab in the serum, suggesting that the precursors for IgA-producing cells are in the brain. Isotype switching of SINV-specific ASCs and/or further maturation of infiltrating naïve B cells and activated IgM⫹ IgD⫺ unswitched B cells could provide local precursors. At 1 month after infection, the expression of MHC class II was decreased on cIgG⫹ B cells, suggesting that these ASCs had undergone further differentiation in the CNS. The late detection of SINV-specific IgA in the bone marrow could be due to the trafficking of these cells from the brain or further differentiation within the bone marrow. Ongoing differentiation of PBs was also observed in the brains of mouse hepatitis virusinfected mice (53). Although the level of viral RNA is substantially reduced by 2 months after infection, it is not eliminated. This was observed previously in BALB/c mice infected with the AR339 strain of SINV (22, 55), so the current study of C57BL/6J mice infected with TE demonstrates that this late pattern of SINV RNA persistence is not restricted to a single mouse or virus strain. Long-term maintenance of antiviral ASCs is essential for continued local Ab production and is likely to be important for the prevention of viral recrudescence. Retention of antiviral ASCs has been observed following other neurotropic virus infections, such as those caused by measles virus (38), West Nile virus (48), rabies virus (17), and mouse hepatitis virus (52, 53). Clinical evidence of the importance of sustained suppression of virus replication in the CNS comes from experience with rituximab (anti-CD20) for the elimination of B cells and with natalizumab (anti-VLA-4) for prevention of the entry of inflammatory cells into the CNS, where a major complication has been reactivation of CNS virus infection (8, 20). In conclusion, the noncytolytic clearance of SINV from neurons during recovery from encephalomyelitis involves several steps and immune effectors. Clearance of infectious virus is associated with infiltration of CD8⫹ T cells and IgM-secreting PBs. This is followed by a period of several weeks during which there is a gradual elimination of viral RNA, associated with the presence of CD4⫹ T cells and IgG- and IgA-secreting PBs that are steadily enriched for cells secreting SINV-specific Abs. Residual low levels of viral RNA are accompanied by the retention of SINV-specific ASCs and the suppression of virus reactivation. The normal function of the blood-brain barrier and the absence of local GC activity indicate that the brain is capable of providing a microenvironment that fosters the longterm maintenance of antiviral ASCs. ACKNOWLEDGMENTS This work was supported by research grant R01 NS038932 and training grant T32 AI007417 from the National Institutes of Health. We thank Patricia Gearhart for helpful discussions, Stephen Nutt (Walter and Eliza Hall Institute of Medical Research) for the generous gift of Blimp-1GFP/⫹ reporter mice, and Cornelia Bergmann (Cleveland Clinic) for shipment of the mice to us.

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