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Journal of Virology, October 2008, p. 9978-9993, Vol. 82, No. 20
0022-538X/08/$08.00+0     doi:10.1128/JVI.01326-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

CD20, CD3, and CD40 Ligand Microclusters Segregate Three-Dimensionally In Vivo at B-Cell-T-Cell Immunological Synapses after Viral Immunity in Primate Brain

Carlos Barcia,^1,2 Aurora Gomez,^1,2 Vicente de Pablos,^1,2 Emiliano Fernández-Villalba,^1,2 Chunyan Liu,³ Kurt M. Kroeger,³ Javier Martín,¹ Andrés Fernández Barreiro,^1,2 Maria G. Castro,^3,4 Pedro R. Lowenstein,^3,4, and Maria-Trinidad Herrero^1,2*,

Clinical and Experimental Neuroscience,¹ Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas, School of Medicine, University of Murcia, Campus de Espinardo, 30100 Murcia, Spain,² Board of Governors’ Gene Therapeutics Research Institute, Cedars-Sinai Medical Center, Los Angeles, California 90048,³ Department of Medicine, and Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California 90095⁴

Received 25 June 2008/ Accepted 28 July 2008

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The clearance of virally infected cells from the brain is mediated by T cells that engage antigen-presenting cells to form supramolecular activation clusters at the immunological synapse. However, after clearance, the T cells persist at the infection site and remain activated locally. In the present work the long-term interactions of immune cells in brains of monkeys were imaged in situ 9 months after the viral inoculation. After viral immunity, the persistent infiltration of T cells and B cells was observed at the infection sites. T cells showed evidence of T-cell receptor signaling as a result of contacts with B cells. Three-dimensional analysis of B-cell-T-cell synapses showed clusters of CD3 in T cells and the segregation of CD20 in B cells, involving the recruitment of CD40 ligand at the interface. These results demonstrate that immunological synapses between B cells and T cells forming three-dimensional microclusters occur in vivo in the central nervous system and suggest that these interactions may be involved in the lymphocyte activation after viral immunity at the original infection site.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The capacity of the immune system to clear viral infections and prepare itself for potential new attacks is still under investigation (for reviews on viral immunity, see references 16 and 26). However, the success of any antiviral response is determined by timing, location, and amount (15), three factors that are crucial for viral immunity since clearance depends on the velocity of the immune response, the type of tissue infected, and the infectivity of the virus. Accordingly, an essential factor for viral immunity is intercellular communication, especially the interaction between lymphocytes and antigen-presenting cells (APC). However, to dissect the complexities of immune intercellular communication, in vivo and in situ imaging of these processes is crucial even though such a task is technically very challenging (23). We previously imaged in vivo the intercellular communication occurring during the clearance of virus-infected cells from the brain (6). As seen from the recruitment and polarization of tyrosine kinases such as Lck and ZAP-70, T cells after activation form supramolecular activation clusters (SMAC) at the immunological synapse, and they mediate the elimination of virus-infected cells (6). SMAC formation in vivo is characterized by a peripheral ring of lymphocyte function-associated antigen 1 (p-SMAC) surrounding a central accumulation of T-cell receptor (TCR) (c-SMAC) at the interface, providing the distinctive bull's-eye structure, as was first described in vitro (31). Interestingly, this interaction between T cells and APC requires the participation of gamma interferon and granzyme B as effector molecules to clear viral infection (7). However, it has been described how, after clearance of virus in the brain, lymphocytes persist in the areas of infection and remain active even if there is no evidence of viral proteins (24, 29, 30). Interestingly, they seem to reside locally and be activated in situ, although it is not known how they remain active. Data indicate that a component of long-term memory may persistently activate cells at the site of initial infection with no evidence of local cellular proliferation (24, 25, 27, 28). This persistency of subsets of memory T cells and B cells after viral immunity could vary depending on the duration and pattern of viral infection in the tissue (26) and also on the interactions between immune cells locally (for reviews, see references 16 and 26). In fact, contact-dependent signals between B and T cells are important for a successful immune response (20). CD40 ligand (CD40L)-CD40 interactions, particularly, are thought to be crucial for the initiation of specific T-cell immune responses (20-22) and for maintaining subsets of memory T-cell populations for viral immunity (9, 12).

In the present work, since B cells are able to function as APC (8, 33, 35) and it is accepted that they play an important role in viral immunity (42) by establishing memory T cells through CD40-CD40L interactions (9, 11, 36, 37), we imaged in vivo the microanatomy of immunological contacts between T cells and B cells in brain sections from monkeys 9 months after adenoviral infection in the brain. Importantly, we observed that B cells and T cells still formed immunological synapses in the brain 9 months after viral infection and that T cells remained activated locally. The fact that B cells and T cells interact locally through CD40L signals, together with CD3 cluster formation and CD20 segregation at the interface of the B-cell-T-cell immunological synapse, suggests that such interactions may be involved in maintaining lymphocyte activation at the original site of infection.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Adenoviral vectors. The adenoviruses used in this study were first-generation E1/E3-deleted recombinant adenovirus vectors based on adenovirus type 5. The construction of Ad-mCMV-βgal (previously named RAd36) (expressing β-galactosidase [β-gal] and containing the murine cytomegalovirus [mCMV] promoter) and Ad-hCMV-HPRT (previously named RAdHPRT) (expressing hypoxanthine-guanine phosphoribosyl-transferase and containing the human CMV [hCMV] promoter) has been described in detail elsewhere (4, 14).

Animals, surgical procedures, and viruses. Seven adult (either male or female) cynomolgus monkeys (Macaca fascicularis) (5-kg body weight) were used for this study, which was carried out according to University of Murcia approved protocols. The animals were injected bilaterally with 1 x 10⁷ infective units of Ad-mCMV-βgal adenovirus in six brain coordinates, including cortex and white matter, as depicted in Fig. 1. All microinjections were performed in a total volume of 1 µl according to previous protocols (4, 41). In order to confirm the infectivity of the virus, at 7 days after intracranial injection one randomly chosen animal was anesthetized by overdose, transcardially perfused-fixed with 2 to 5 liters of saline solution, and then perfused with 4% paraformaldehyde to fix the tissue. The brain was removed and sectioned with a microtome (Microm) into 50-µm sections.

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FIG. 1. Infection of Ad-mCMV-βgal adenovirus induces expression of β-gal in monkey brain astrocytes and induces MHC molecule expression. (A) Schematic diagrams of the anatomical positions of stereotaxic microinjections of Ad-mCMV-βgal adenovirus performed in monkey brain. The left panel represents a view of macaque brain, where each colored circle represents a microinjection at assigned coordinates (red circles, right hemisphere; green circles, left hemisphere). The right panel represents a coronal section of the brain at one of the coordinates. The red line (right hemisphere) and green line (left hemisphere) represent the injection sites in the cortex. (B) β-Gal is expressed in astrocytes (GFAP-positive cells) but not in neurons (MAP-2-positive cells). Confocal pictures show two cells infected with Ad-mCMV-βgal adenoviral vector (expressing β-gal) combined with GFAP staining for astrocytes, MAP-2 staining for neurons, and DAPI for counterstaining. β-Gal-positive cells colocalize with GFAP staining but not with MAP-2 staining. Bar, 50 µm. (C) Seven days after Ad-mCMV-βgal infection, monkey brain sections show β-gal-positive cells, as seen by immunohistochemistry at the injection sites. Adjacent sections also showed HLA-DR expression by immunohistochemistry around the injection site. Left column, lower magnification (bar, 5 mm); right column, higher magnification (bar, 250 µm).

The other six monkeys were used for long-term analysis. To ascertain whether peripheral viral rechallenge affects the persistence of the lymphocyte population and cell-cell interactions 2 months later, the remaining animals were anesthetized briefly and injected intradermally in the back with 100 µl of either sterile saline (n = 3, nonrechallenged) or 1 x 10⁸ infectious units of Ad-hCMV-HPRT adenovirus (n = 3, rechallenged). Seven months later (9 months after brain infection), the six animals were anesthetized by overdose and transcardially perfused-fixed with 2 to 5 liters of saline solution. Immediately afterwards, the animals were perfused with 4% paraformaldehyde to fix the tissue, and brains were removed and sectioned with a microtome (Microm) into 50-µm sections.

Immunohistochemical procedures. The 50-µm coronal brain sections were cut serially through the entire brain, and diaminobenzidine detection or immunofluorescence was performed as described previously (4, 6, 41), using primary antibodies to recognize CD3 (1:100, rabbit; Dako), CD20 (1:500, mouse immunoglobulin G2a [IgG2a]; Dako), β-gal (1:1,000, rabbit mouse IgG; Promega), HLA-DR (1:50, mouse IgG2a; Dako), microtubule-associated protein 2 (MAP-2) (1:500, chicken; Abcam), myelin basic protein (1:100, rat IgG2b; Chemicon), glial fibrillary acidic protein (GFAP) (1:500, guinea pig; Advanced Immunochemical), phosphorylated ZAP-70 (p-ZAP-70) (1:50, rabbit; Cell Signaling), CCL2 (MCP-1) (1:40, mouse IgG2b; R&D), CD45RO (UCHL1) (1:30, mouse IgG2a; Ventana), CD11b (1:100, rat IgG2b; Abcam), and CD40L (1:100, goat; R&D).

For diaminobenzidine staining, endogenous peroxidase activity was quenched with 0.3% H₂O₂ in phosphate-buffered saline (PBS), nonspecific Fc-binding sites were blocked with 10% horse serum, and sections were incubated for 48 h at room temperature with primary antibody diluted in PBS containing 1% horse serum and 0.5% Triton X-100 (antibody solution). Sections were then incubated for 4 h with appropriate biotin-conjugated secondary antibodies (Dako, Cambridge, United Kingdom). Antibody binding was detected using avidin-biotin peroxidase with diaminobenzidine as a chromogen. These sections were mounted on gelatinized glass slides and dehydrated before coverslipping.

For immunofluorescence, a series of sections of each brain were pretreated with 10 mM citrate buffer, pH 6, for 30 min at 65°C in order to increase antigen retrieval and penetration of the antibodies into the tissues. Sections were blocked with 1% Triton X-100 for 5 min and with 3% normal horse serum in 0.1 M PBS, pH 7.4, for 60 min. Sections were incubated at room temperature for 48 h with combined primary antibodies. For multiple staining, incubation with primary antibodies was followed by 4 h of incubation with the appropriate secondary antibodies (Alexa 488, Alexa 546, Alexa 594, and/or Alexa 647 [1:1,000; Molecular Probes]).

After washing, sections were incubated with DAPI (4',6'-diamidino-2-phenylindole) solution for 30 min. Sections were washed, mounted, examined by conventional fluorescence microscopy (Axiolab; Zeiss), and analyzed with a confocal microscope (DMIRE2; Leica Microsystems, Exton, PA).

Stereological quantification. The number of cells was estimated in a blinded manner by stereological methods at the injection sites using the optical fractionator probe as described previously (5). The area of quantification was the rectangular area enclosing the injection sites (5 mm by 10 mm). Positive cells were quantified using unbiased stereological methods (38) with a computer-assisted image analysis system (ScionImage) and a Zeiss microscope connected to a digital camera (CoolSnap) through a Zeiss zoom set at 12.5x and a 0.1x adapter. The number of cells was measured in 250-µm-side squares (dissectors), 750 µm (x) and 750 µm (y) apart, systematically covering the whole surface areas of the analyzed regions. Approximately 50 fields were quantified per section. Using the principle of the optical dissector, positive cells were counted only when they cut the top and left-hand borders of the square, as previously described (38). Results are expressed as an estimation of the absolute number of positive cells in the injection sites analyzed, since the thickness of the sections and the number of the series of sections were considered.

Confocal analysis. Brain sections were examined using a Leica DMIRE2 confocal microscope with the 63x oil objective and Leica confocal software (Leica Microsystems Heidelberg 19 GmbH). A series range for each section was set by determining upper and lower thresholds using the Z/Y Position for Spatial Image Series setting, and confocal microscope settings were established and maintained by Leica and local technicians for optimal resolution. (See references 6 and 7 for further details on in vivo imaging of immunological synapses.) Contacts were defined as areas where colocalization of both markers occurs between two cells in at least two 0.5-µm-thick optical sections. Images can also be illustrated as they appear throughout the stack of sections as a simple 0.5-µm layer or as a transparency of all layers merged together.

Confocal quantifications. The percentages of synapsing cells were quantified using confocal microscopy (CD3-CD11b, CD3-CD20, and CD20-CD11b). Images containing positive cells were randomly captured, covering the injection sites in both groups of animals. In each stack of images, the number of positive cells and the number of cells engaged in synapses were counted with the Leica confocal software, and the percentage of cells engaged in synapse compared to the total number of cells was calculated.

After the quantification, some randomly found synapses were captured in high resolution and analyzed in close detail in order to observe the pattern of distribution of the CD3, CD20, and CD40L molecules in the region of intercellular contact and along the membrane. By means of the Leica confocal software, relative fluorescence intensity was quantified along the membrane in a single 0.5-µm optical section from the z stack at the center of the immunological synaptic interface and is illustrated in the figures with corresponding arrows traversing the measured optical planes. The frequency of clusters and rings at the interface was also quantified. Then, three-dimensional reconstructions at the interface were rendered with alpha blending software (Imaris; Bitplane AG), which allowed the free rotation of the stack of images and observation of how the molecules were distributed at the interface. This three-dimensional analysis, together with the relative fluorescence analysis, permitted the type of synapses to be identified from the ring or cluster formation. The total number of synapses studied in detail with the confocal microscope in this study was 104 (39 synapses for CD20/CD3, 21 for CD20/p-ZAP-70, and 44 for CD20/CD3/CD40L).

Note that given the complexity of the confocal analysis, the total number of immunological synapses illustrated throughout this paper in detail demonstrates the existence of B-T immunological synapses in vivo but obviously cannot be considered a completely faithful estimation of their total number. Currently, it remains technically impossible to record accurately the exact number of mature immunological synapses present in vivo.

Statistical analysis. Viability data were expressed as mean ± standard error of the mean and evaluated by two- or one-way analysis of variance (followed by Dunnet or Tukey multiple-comparison tests) or Student t test. Differences were considered significant if the P value was <0.05. Pearson's correlation value was also used for correlation between variables.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Infection of the adenovirus Ad-mCMV-βgal induces β-gal expression in astrocytes in the brain of Macaca fascicularis. Seven monkeys were microinjected intracranially with 10⁷ infective units of Ad-mCMV-βgal adenovirus, as described in Materials and Methods (Fig. 1A). In order to confirm the infectivity of Ad-mCMV-βgal adenovirus, one of the animals was sacrificed 7 days after intracranial injection, and its brain was removed and analyzed. Adenovirus-infected cells were stained with β-gal immunohistochemistry at the injection sites (Fig. 1B and C). Confocal analysis of the multiple immunofluorescence staining demonstrated that infected (β-gal-expressing) cells were astrocytes (GFAP-positive cells) (Fig. 1B) but not neurons (MAP-positive cells). Furthermore, HLA-DR immunostaining of adjacent sections demonstrated the expression of major histocompatibility complex (MHC) class II molecules around Ad-mCMV-βgal injection sites (Fig. 1C).

Immune-mediated clearance of adenovirus-infected cells causes persistency of lymphocytes. The rest of the animals (n = 6) were used to study the persistency of the lymphocytes after immune-mediated viral clearance. In order to demonstrate whether peripheral viral rechallenge induces changes in the final population of persistent lymphocyte subsets or modifies their intercellular interactions, three of the monkeys were rechallenged systemically with Ad-hCMV-HPRT adenovirus 2 months after surgery. The other three were injected with saline. Nine months after the intracranial injection, the monkeys were sacrificed, and the brains were then removed and analyzed in detail. After 9 months, no evidence of adenovirus-infected cells (expressing β-gal) was found in any of the animals at any injection site (rechallenged or not) (Fig. 2A). Furthermore, a massive and chronic infiltration of CD3⁺ T cells and CD20⁺ B cells was observed in the brain at the microinjection sites in parenchyma and around blood vessels in both groups of animals (Fig. 2B). Infiltration was mostly evident in the injection sites, but some lymphocytes were also seen along the corpus callosum, close to the areas of injection, and in the septum, subarachnoid space, and subventricular areas. At the viral injection sites, blood vessels showed large amounts of CD3⁺ T cells and CD20⁺ B cells (Fig. 2B). However, in the brain parenchyma the number of infiltrated CD20⁺ B cells was lower than that of CD3⁺ T cells (approximately a 1:4 ratio). Interestingly, rechallenged monkeys showed a statistically significant increase in CD20⁺ B-cell number and a higher number of CD3⁺ T cells in the injection sites compared with nonrechallenged animals (Fig. 2C). To ascertain whether the infiltrated T cells were memory T cells, we stained adjacent series of sections, combining CD3 and CD45RO. We observed CD3⁺/CD45RO⁺ T cells in the injection sites around blood vessels. When the proportions of infiltrated memory T cells in both groups were quantified and compared, CD3⁺/CD45RO⁺ T cells were found to be more numerous in the injection site in the rechallenged animals (Fig. 2D). Furthermore, no evident demyelination was found in white matter around the injection sites (data not shown).

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FIG. 2. Nine months after viral infection, a high infiltration of lymphocytes persists at the injection sites. (A) Schematic diagram of a coronal macaque brain section. The red rectangle shows the anatomical area of the microinjections magnified on the right. Sections of monkey brain were stained for β-gal at 7 days and 9 months after intracranial adenoviral infection. The pictures show that 7 days after the injection, infected astrocytes (β-gal positive) can be seen by immunohistochemistry. However, 9 months after intracranial infection, the immune system has cleared infected cells in both rechallenged and non rechallenged animals. Bar: 1 mm. (B) The confocal picture shows infiltration of T cells (CD3+ cells in green) and B cells (CD20+ cells in red) through a blood vessel at the injection site of a monkey at 9 months after intracranial adenoviral infection. (C) The graph shows the stereological estimation of the number of T cells (CD3+ cells) and B cells (CD20+ cells) infiltrated in the brain parenchyma of monkeys infected intracranially with adenovirus (rechallenged or nonrechallenged). Rechallenged animals showed a higher number of infiltrated CD3+ T cells and a significant increase in their CD20+ B-cell population. Pictures of CD3+ and CD20+ cells in monkey brain parenchyma are also shown. (D) Quantification of CD3+/CD45RO+ T cells (memory T cells) and CD3+/CD45RO– cells in rechallenged and nonrechallenged animals at the injection sites. Memory T cells (CD3+/CD45RO+) were more abundant in rechallenged animals. The pictures on the right show confocal pictures of the two cell types quantified (CD3+/CD45RO+ in the top row and CD3+/CD45RO– in the bottom row).

Because of the high degree of lymphocyte infiltration located in the injection sites, we analyzed the expression of the chemokine CCL2, which is responsible for T-cell and macrophage infiltration in the parenchyma, by immunohistochemistry to ascertain whether its expression retained the lymphocytes locally. Immmunostaining demonstrated that only the areas of injection, in both rechallenged and non rechallenged animals, showed a high concentration of CCL2-expressing cells that were not found in noninjected areas (Fig. 3).

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FIG. 3. CCL2 is expressed in the injection sites at 9 months after viral injection. Since T-cell infiltration was observed, we analyzed the expression of the chemokine CCL2 in the areas of infiltration by immunohistochemistry. (A) Schematic diagram of the anatomical localization of the stereotaxic microinjections of Ad-mCMV-βgal adenovirus in monkey brain. (B) Representative section of an injected cortex (a, a', and a'') compared with a noninjected cortex (b, b', and b''). The region indicated by the arrow is shown at higher magnification in panel a', and a detail of CCL2-positive cells is shown in panel a''. All injection sites in both groups of animals showed CCL2 expression. No CCL2-positive cells were detected in noninjected areas. Bars: a and b, 2 mm; a' and b', 500 µm; a'' and b'', 150 µm.

Lymphocytes are engaged in cell-cell interactions in the brain parenchyma. Since we observed infiltrating lymphocytes locally at the injection sites, the intercellular interactions were analyzed in detail. Study of the intercellular interactions between T cells, B cells, and microglia/macrophages showed that CD3⁺ T cells were in contact with CD11b⁺ microglia/macrophages or with CD20⁺ B cells in the brain parenchyma (Fig. 4). In the same way, CD20⁺ B cells were seen in contact with CD11b⁺ microglia/macrophages or with CD3⁺ T cells (Fig. 4). Furthermore, rechallenged animals showed a significant increase in the percentage of T cells establishing immunological synapse with B cells in the brain parenchyma compared with nonrechallenged animals (Fig. 4). In contrast, no changes were found in the percentage of B cells establishing synapses with T cells or with microglia/macrophages (Fig. 4).

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FIG. 4. Rechallenged animals show a higher percentage of T cells establishing synapses with B cells. Double immunofluorescence of CD3/CD11b, CD3/CD20, or CD20/CD11b in the brain sections was performed, and the proportion of cells engaged in synapses was quantified by confocal microscopy. The pictures show a representative example of each type of synapse quantified. The channels for the different staining, combined with DAPI as a counterstain, are shown in each row. The first row of images illustrates a CD3+ T cell engaged in synapse with a CD11b+ cell (microglia/macrophage). The second row shows a CD3+ T cell engaged in synapse with a CD20+ B cell, and the third row illustrates a B cell engaged in synapse with a CD11b+ cell (microglia/macrophage). The top pie graphs show the percentage of T cells engaged in synapses with B cells (red) or with microglia/macrophages (gray) in both nonrechallenged and rechallenged animals. The bottom pie graphs show the percentage of B cells synapsing with T cells (green) or with microglia/macrophages (gray). There was a significant increase in the number of CD3+ T cells establishing synapses with B cells in rechallenged monkeys compared with nonrechallenged animals (10% versus 2%). No differences were found in the rest of synapses quantified.

Activation of T cells 9 months after viral immunity. T-cell activation was assessed from the expression of p-ZAP-70 (recognizing pY319), a tyrosine-kinase specific for T-cell activation after TCR signaling (6, 10). Both groups of monkeys (rechallenged and nonrechallenged) showed p-ZAP-70-positive T cells at the injection sites (Fig. 5A), although all three rechallenged monkeys showed similar numbers of p-ZAP-70 cells at the injection sites. Only one of the nonrechallenged animals showed high numbers of activated T cells, while the others showed very low numbers (Fig. 5A). Most importantly, detailed confocal analysis revealed that p-ZAP-70-positive T cells were found in contact with B cells (Fig. 5B), suggesting T-cell activation as a result of the interaction with B cells. Most importantly, there was a strong correlation between the number of activated T cells and the number of B-cell-T-cell synapses formed at the injection sites (Fig. 5C).

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FIG. 5. Activated T cells are present in monkey brain parenchyma at 9 months after intracranial infection and are found in contact with B cells. (A) Activated T cells (p-ZAP-70+ positive) were quantified at the injection sites 9 months after intracranial infection in both rechallenged and nonrechallenged animals. The graph shows that rechallenged animals normally have a larger population of p-ZAP-70-positive cells at the injection sites (although no statistically significant changes between the groups were observed). Bar, 50 µm. (B) Stainings of CD20 for B cells (red), p-ZAP-70 (green) for activated T cells, and DAPI (blue) (as counterstaining) were combined. Detailed confocal analysis showed p-ZAP-70-positive cells in contact with CD20+ B cells. The panels show three synapses between B cells and p-ZAP-70-positive T cells. Synapses 1 and 3 show some degree of polarization, with larger amounts of p-ZAP-70 at the cellular pole in contact with the B cell and very low p-ZAP-70 levels at the opposite pole. Synapse 2 displays p-ZAP-70 clustered at the center of the interface. In the three cases shown, p-ZAP-70 is somehow polarized toward the synapse interface. All three synapses were obtained from rechallenged animals. (C) Correlation between the number of activated T cells and the number of B-cell-T-cell synapses in each monkey 9 months after viral inoculation. The graph was generated by correlating the estimation of the number of activated T cells (expressing p-ZAP-70) on the x axis and the estimation of the total number of B-cell-T-cell synapses on the y axis. The number of B-cell-T-cell synapses was calculated from the stereological estimation of the total number of T cells multiplied by the estimated proportion of B-cell-T-cell synapses. The plot reveals a strong positive relationship between the number of T cells establishing synapses and activated T cells. The value of Pearson's correlation coefficient (r) is 0.75, and the P value is 0.02.

B-cell-T-cell immunological synapses show CD20 and CD3 microclusters. CD3⁺ T cells in contact with CD20⁺ B cells showed a particular distribution of CD3 molecules at the membrane: segregated to the peripheral area of the interface or forming a central cluster (Fig. 6 to 9). Other synapses did not show a well-defined distribution (data not shown). Three-dimensional reconstructions at the interface confirmed that CD3 may be displayed in the form of a ring (p-SMAC) (Fig. 6 and 9) or as a central cluster (c-SMAC) (Fig. 7 and 9). These results confirm in vivo the CD3/TCR dynamics previously observed in vitro, where CD3/TCR p-SMAC precedes CD3/TCR c-SMAC (45).

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FIG. 6. CD3 forms peripheral clusters at B-cell-T-cell immunological synapses, and CD20 is segregated peripherally. Detailed confocal analysis of immunological synapses between B cells (CD20+, in red) and T cells (CD3+, in green) in monkey brain sections was performed. The panels show immunological synapses between T cells and B cells. Fluorescence channels for CD3, CD20, and their merging are shown (A, E, and I). Relative fluorescence was measured along the CD20+ B-cell membrane and synapse interface, as depicted by yellow circular arrows (B, F, and J). The interface is indicated by arrows 1, 2, and 3 in the pictures and relative fluorescence graphs (D, H, and L). Results of the measurements are shown in the graphs. Three-dimensional reconstructions were performed at the interface level as indicated with the orange arrow on the gray scale image (C, G, and K). The reconstructions are shown in fluorescence channels for CD3, CD20, and merge. In all three synapses shown, higher CD20 relative fluorescence is seen at the periphery of the interface (indicated by arrows 1 and 3 in panels B, D, F, H, J, and L). At the center of the interface CD20 relative fluorescence levels were lower (indicated by arrows 2). In all three cases shown, three-dimensional reconstructions revealed that the CD20 molecule has a ring-shaped distribution at the interface. However, the relative fluorescence for CD3 in the three synapses reveals a pattern with maximum fluorescence in the peripheral area (indicated by arrows 1 and 3) and minimum fluorescence at the center of the interface (indicated by arrows 2). Three-dimensional reconstructions showed that CD3 forms a ring shape similar to that of CD20.

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FIG. 9. CD3 in T cells engaged in synapses with B cells polarizes to the synaptic interface. (A) Measurements of the relative fluorescence of CD3 along the membranes of T cells not engaged in synapses with B cells reveal an irregular pattern with no particular polarization. Three nonsynapsing T cells (a, b, and c) are shown. (B) Synapsing T cells (obtained from B-cell-T-cell synapses) with their relative fluorescence measurements. T cells in panels d, e, and f (cluster type) are T cells that show a central cluster of CD3, with the maximum of relative fluorescence at the center of the interface (arrows). T cells in panels g, h, and i (ring type) show segregation of CD3 to the peripheral area of the interface with two maxima of relative fluorescence at the periphery (arrows in panels g, h, and i). The T cells in panels d and e are also shown in Fig. 7. The T cell in panel f is also shown in Fig. 11. The T cells in panels h and i are also shown in Fig. 6.

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FIG. 7. CD3 forms central clusters at B-cell-T-cell immunological synapses, and CD20 is segregated peripherally. Detailed confocal analysis of immunological synapses between B cells (CD20+, in red) and T cells (CD3+, in green) in monkey brain sections was performed. Panels show immunological synapses between T cells and B cells. Fluorescence channels for CD3, CD20, and their merging are shown (A, E, and I). Relative fluorescence was measured along the CD20+ B-cell membrane and synapse interface, as depicted by yellow circular arrows (B, F, and J). The interface is indicated by arrows 1, 2, and 3 in the pictures and relative fluorescence graphs (D, H, and L). Results of the measurements are shown in the graphs. Three-dimensional reconstructions were performed at the interface level as indicated with the orange arrow on the gray scale image (C, G, and K). The reconstructions are shown in fluorescence channels for CD3, CD20, and merge. In all three synapses shown, higher CD20 relative fluorescence is seen at the periphery of the interface (indicated by arrows 1 and 3 in panels B, D, F, H, J, and L). At the center of the interface, CD20 relative fluorescence levels were lower (indicated by arrows 2). In all three cases shown, three-dimensional reconstructions revealed that the CD20 molecule has a ring-shaped distribution at the interface. However, relative fluorescence for CD3 was maximum in the central area (indicated by arrows 2) and minimum at the periphery of the interface (indicated by arrows 1 and 2). Three-dimensional reconstructions revealed that CD3 forms a central cluster.

CD20⁺ B cells also show a particular microanatomy at the interface. To analyze the CD20 distribution, levels of relative fluorescence were measured at the interface. Most frequently, higher values of CD20 relative fluorescence were observed at the periphery of the interface (Fig. 6 and 8), while lower levels of CD20 fluorescence were observed at the center of the interface where CD3 was present (Fig. 6 and 8). Three-dimensional reconstructions at the interface of these synapses demonstrate that CD20 is distributed specifically at the interface of B-cell-T-cell synapses, forming a p-SMAC-like ring and segregating CD20 molecules to the peripheral area of the interface (Fig. 6 and 8). CD20 molecules were rarely observed in the central area of the interface (see Fig. 12). This CD20 p-SMAC structure surrounded a central area at the interface whether CD3 formed a ring or a central cluster (Fig. 6 and 7). Synapses 1, 2, 3, and 6 shown in Fig. 6 to 8 were captured from nonrechallenged animals, and synapses 4, 5, 7, 8, and 9 shown in Fig. 6 to 8 were captured from rechallenged animals. B cells not in contact with T cells did not show any particular distribution (see Fig. 13).

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FIG. 8. CD20 is segregated peripherally even if CD3 does not form clusters at B-cell-T-cell immunological synapses. The panels show immunological synapses between T cells and B cells. Fluorescence channels for CD3, CD20, and their merging are shown (A, E, and I). Relative fluorescence was measured along the CD20+ B-cell membrane and synapse interface, as is depicted by yellow circular arrows (B, F, and J). The interface is indicated by arrows 1, 2, and 3 in the pictures and relative fluorescence graphs (D, H, and L). Results of the measurements are shown in the graphs. Three-dimensional reconstructions were performed at the interface level as indicated with the orange arrow on the gray scale image (C, G, and K). The reconstructions are shown in fluorescence channels for CD3, CD20, and merge. In all three synapses shown, higher CD20 relative fluorescence was seen at the periphery of the interface (indicated by arrows 1 and 3 in panels B, D, F, H, J, and L). At the center of the interface, CD20 relative fluorescence levels were again lower (indicated by arrows 2). In all three cases shown, three-dimensional reconstructions revealed that the CD20 molecule has a ring-shaped distribution at the interface even if the relative fluorescence measurements and the three-dimensional reconstructions of CD3 did not reveal a well-defined arrangement at the synapse interface.

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FIG. 12. Confocal analysis of B-cell-T-cell synapses reveals some B cells where CD20 forms a central cluster. As in Fig. 10 and with a similar organization, two synapses are shown in detail. The fluorescence channels of each synapse are shown in panels A and F. Relative fluorescence measurements revealed that CD40L and CD20 are clustered at the center of the synapse (panels B and G for T cells and panels C and H for B cells). Three-dimensional reconstructions revealed that CD40L and CD20 appear to form central clusters (E and J). Schematic representations of the synapses are depicted in panels D and I.

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FIG. 13. Nonsynapsing T cells do not show a CD40L cluster, while nonsynapisng B cells do not show CD20 segregation. (A) Relative fluorescence measurements of CD40L in three nonsynapsing T cells show very low levels of fluorescence and no specific pattern of distribution. (B) Relative fluorescence measurements of CD20 in three nonsynapsing B cells do not show a specific pattern of distribution.

CD40L is recruited at the B-cell-T-cell synapse interface. Confocal imaging of immunological synapses between B cells and T cells revealed that CD40L is specifically recruited at the interface (Fig. 10 to 12) with a specific distribution. To analyze the distribution of CD40L, measurements of relative fluorescence were made at the membranes of T cells and B cells. Maximum values of CD40L were observed at the interface of the synapse between T and B cells. Some of the synapses showed higher CD40L fluorescence at the center of the interface (Fig. 10 and 12). However, other synapses revealed maximum CD40L values at the periphery of the interface (Fig. 11). Three-dimensional reconstructions revealed that CD40L can be clustered at the center of the interface or forming a ring-shaped structure (Fig. 10 to 12). Interestingly, the three-dimensional reconstructions also revealed that clustered CD40L can be observed coinciding with CD3 clusters and that they were surrounded by a CD20 peripheral ring (Fig. 10). Three-dimensional reconstructions also showed that when CD40L appeared in a ring shape, it coincided with a CD20 ring (Fig. 11). Synapses 1, 2, 3, 6, and 8 in Fig. 10 to 12 were captured from rechallenged animals, and synapses 4 and 5 in Fig. 10 to 12 were captured from nonrechallenged animals. T cells not in contact with B cells show CD40L in the cytoplasm and with no particular polarization (Fig. 13).

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FIG. 10. The CD40L recruited at the B-cell-T-cell immunological synapse forms a central cluster. B-cell-T-cell immunological synapses were analyzed in detail by confocal microscopy, combining CD20 (red), CD3 (green), CD40L (magenta) and DAPI (blue) as counterstaining. Three synapses are shown in detail. DAPI, CD3, CD20, and CD40L channels, as well as the merged channels CD40L/DAPI, merge 1 (CD3/CD20/CD40L), and merge 2 (CD3/CD20/CD40L/DAPI), are shown in panels A, F, and K. The relative fluorescence of each T-cell and B-cell forming synapses was measured along the membrane as is indicated in the gray scale images by circular arrows in panels B, G, and L for T cells and panels C, H, and M for B cells. The interface is indicated by arrows 1, 2, and 3. Measurements of relative fluorescence are shown in the graphs. Schematic representations of the synapses are depicted in panels D, I, and N. Three-dimensional reconstructions of the interface are shown in panels E, J, and O. In the three synapses studied, relative fluorescence of CD40L increases at the B-cell-T-cell interface (indicated between arrows 1 and 3). In these three cases, the highest relative fluorescence was observed at the center of the interface (arrows 2) and the lowest in the peripheral region (arrows 1 and 3). Three-dimensional reconstructions at the interface revealed that CD40L forms a central cluster surrounded by a CD20 ring.

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FIG. 11. CD40L can also be found segregated to the periphery of the interface. As in Fig. 10 and organized in the same way, three synapses are shown in detail. The fluorescence channels of each synapse are shown in panels A, F, and K. Relative fluorescence measurements revealed that CD40L is segregated to the periphery (panels B, G, and L for T cells and panels C, H, and M for B cells). Three-dimensional reconstructions revealed that CD40L appears to be segregated to the periphery, forming a ring-shaped cluster (E, J, and O). Schematic representations of the synapses are depicted in panels D, I, and N.

Finally, in order to determine the frequency of the arrangements in rechallenged and nonrechallenged animals, we quantified the percentage of arrangements found in the B-cell-T-cell synapses, observing that B-cell-T-cell interactions were more frequent in rechallenged animals than in nonrechallenged animals (Fig. 14A). It was also seen that the percentage of clustering is increased at the B-cell-T-cell interface in rechallenged animals, except in regard to the formation of the CD3 central cluster (Fig. 14B).

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FIG. 14. Rechallenging induces changes of the cluster formation in B-cell-T-cell synapses in vivo in monkey brain after viral immunity. (A) Quantification of the total number of synapses found and studied in detail in rechallenged and nonrechallenged animals. Rechallenged animals showed a higher number of synapses than nonrechallenged ones. (B) Percentages of B-cell-T-cell synapses forming different structures at the interface with respect to the total number of synapses studied. The percentage of cluster formation was higher for rechallenged animals in each case, except for the CD3 cluster. The formation of a CD20 ring was the most frequent structure at the interface, while a CD20 central cluster was very rare. (C) Two B-cell-T-cell synapses with the most frequent arrangements of the interface observed in the monkey brain in vivo after viral immunity. The top illustration depicts a B-cell-T-cell synapse forming a central cluster of CD3 and CD40L at the interface with segregation of CD20 to the periphery. The bottom illustration shows a B-cell-T-cell synapse forming a peripheral cluster (ring-like) of CD3 and CD40L with CD20 segregated toward the periphery.

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In the present work we observed that B cells and T cells engaged in an immunological synapse in the brain in vivo. Furthermore, this intercellular communication between B cells and T cells implies the segregation of different molecules at the interface of the immunological synapse. Previous and very striking in vivo images of B-cell-T-cell interactions in the germinal centers of lymph nodes using two-photon microscopy demonstrated the dynamics of these cell-cell connections (1, 2). However, the two-photon microscope still does not provide sufficient resolution to discriminate microscopic formations such as SMAC-like arrangements. In the present work, we demonstrate that B-cell-T-cell interactions occur outside the lymphoid organs and with a particular microanatomy.

First, CD3 is clustered at the B-cell-T-cell interface. Three-dimensional reconstructions demonstrated that CD3 may move to the periphery of the interface, forming a p-SMAC (Non-Kupfer-type synapse) (Fig. 6 and 9), or be recruited to the central region of the interface, forming a c-SMAC (Kupfer-type synapse) (Fig. 7 and 9). When the frequency of CD3 rearrangements at B-cell-T-cell synapses was quantified, rechallenged animals showed a higher percentage of CD3 ring formation than nonrechallenged animals. However, the CD3 central cluster was the same in both groups of animals (Fig. 14). These two appearances have been described in vitro as different phases of CD3/TCR clustering at immunological synapses (45). CD3/TCR is first segregated to the periphery before it forms clusters in the center, which reflects the MHC dynamics observed at the interface (19). These results are the in vivo confirmation of CD3/TCR dynamics at the B-cell-T-cell synapse in the context of lymphocyte activation after viral immunity in primate brain.

Second, CD20 is segregated in B cells to form a ring-like structure as a peripheral cluster. This cluster is observed in B cells even though CD3 molecules appear segregated in p-SMAC or c-SMAC in T cells (Fig. 6 to 9 and 14). Very rarely, CD20 molecules were found as a central cluster (Fig. 12 and 14). The function of the CD20 molecule is not completely understood, although it is known that it functions as a Ca²⁺ channel during B-cell activation (13, 39, 40, 43). This feature is consistent with local activation of B cells at the interface, which requires Ca²⁺ accumulation at the immunological synapse, as was previously demonstrated (11), and may be facilitated by CD20 accumulation at the periphery of the interface. Interestingly, the CD20 ring shape can be observed independently of whether CD3 and CD40L present a peripheral or central distribution (Fig. 6 to 8), suggesting that CD20 is a stable long-term structure at the B-cell-T-cell synapse, in accordance with the possible long-term Ca²⁺ demands for B cells.

Finally, this B-cell-T-cell interaction is also characterized by the recruitment of CD40L at the interface (Fig. 10 to 12), a recruitment that is quite evident from the intensity of CD40L relative fluorescence at the interface, which is more than 100 times higher than the basal fluorescence levels measured in the rest of the membrane (Fig. 10 to 12). Importantly, CD40L is observed not only in central cluster but also segregated to the periphery of the interface, forming a ring shape. The quantification of the frequency of rearrangements revealed that rechallenged animals show a higher percentage of B-cell-T-cell synapses forming peripheral and central cluster of CD40L (Fig. 14). The two arrangements seem to be different reflections of the same process and could be equivalent to that described for CD3/TCR clustering (45). In addition, it has been described how, in vitro, CD3/TCR and CD40L are recruited and segregated at the interface in a sequenced manner: CD3/TCR is segregated before, preceding CD40L recruitment, while CD3 clustering is faster and less stable than CD40L recruitment (11), which is coherent with our quantification of the frequency of rearrangements (Fig. 14). CD40L can be observed in CD3⁺ T cells but also in some CD20⁺ B cells (Fig. 10 to 12). Both T cells and B cells are able to express CD40L in their membranes, as was previously described (44, 47). Although it is known that CD40L is expressed in B cells at very low levels, its function is under discussion (47). It is accepted that the CD40L of T cells binds CD40 in B cells. Accordingly, T cells with no apparent immunological synapse engagement show CD40L in the membrane but with no particular distribution or polarization (Fig. 13). It is possible that CD40L was released as a soluble molecule by T cells in the intersynaptic space, binding CD40 at the B-cell membrane (18). In fact, our images show a very high concentration of CD40L at the interface, probably following a mirror image of CD40 at the B-cell membrane.

Quantification of the frequencies of structures formed at the B-cell-T-cell immunological interface suggests that the CD20 ring is a very stable structure whose formation is increased in rechallenged animals. In regard to T cells, the frequency of peripheral clustering of CD3 and CD40L formation appeared to be higher in rechallenged animals. However, the CD40L central cluster was the most frequent structure found and was more abundant with adenoviral rechallenge, in contrast to the case for the CD3 central cluster, which does not vary with intraperitoneal rechallenging (Fig. 14). These results suggest that rechallenging, as well as increasing the frequency of B-cell-T-cell synapses, increases the recruitment of CD40L at the interface and the segregation of CD3 at the periphery.

B-cell-T-cell synapse function is still unclear. It is possible that B cells facilitate the activity of the T cells, or vice versa. It is known that the binding of T-cell CD40L to CD40 in B cells mediates T-cell help (17, 36). It has been demonstrated that B cells are able to prime naïve T cells in vivo in a CD40L contact-dependent signal (20, 35), which plays a crucial role in viral immunity (12, 20). Previous studies reported that adoptively transferred antigen-specific T cells lacking CD40L failed to expand upon antigen challenge of the recipients, demonstrating that the expression of CD40L in T cells is required for the in vivo priming of T cells and therefore for the initiation of specific T-cell immune responses (21, 22). Therefore, the presence of CD40L in T cells and its engagement with B cells could be important for clearance of viral infection. Consequently, and in accordance with previous reports, these CD40L-dependent signals could help maximize effector or memory T cells and contribute to their local long-term maintenance after viral infection (26, 32, 34, 46). It is also possible that CD40L interactions are responsible for T-cell-dependent B-cell activation, Ig class switching, and the generation of B-cell memory (3, 17). The CD40L signal is crucial for B-cell responses, and the expression in T cells induces clonal expansion of B cells that specifically bind to foreign antigens (20). Therefore, the local CD40L-dependent B-cell-T-cell synapses that we imaged in vivo at the infection sites could be the long-term renewable mechanism that induces lymphocyte activation (effector or memory T cells or B cells) at the original site of viral infection.

On the other hand, as a complete viral clearance was not fully demonstrated, it is plausible that residual virus remains at the injection sites and that what we are observing is a sustained effector response. It is also possible that the viral clearance was higher in rechallenged animals, since we observed an increasing proportion of memory T cells compared with that in nonrechallenged animals.

In conclusion, our results demonstrate that B-cell-T-cell interactions occur in the brain in vivo and that these interactions present a particular microanatomy, with rearrangements of CD20, CD3, and CD40L, which could be involved in the local activation of lymphocytes at the infection sites. Further studies of these cell-cell interactions will help provide new therapeutic targets for modifying local immune responses such as CD40L-dependent B-cell-T-cell immunological synapses. Meanwhile, imaging in vivo and in situ of the microanatomy and molecular distribution involved in the intercellular communication is a central task for unraveling the mechanisms of the immune system and for discerning new biological targets for use in more accurate therapeutic strategies.

 
This work has been supported by grants (to M.-T. Herrero) from the Spanish Ministry of Science (SAF 2004 07656 C02-02), Fundación Séneca (FS/05662/PI/07), and CIBERNED (Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas).

We thank all the personnel from SAI (Servicio de Apoyo a la Investigación), especially María García, for the help provided at the University of Murcia Microscopy Core. We also thank P. Thomas for comments on the manuscript and language suggestions.

We declare that there are no conflicts of interest in the present work.

 
* Corresponding author. Mailing address: Clinical and Experimental Neuroscience, CIBERNED, School of Medicine, University of Murcia, Campus de Espinardo, 30100 Murcia, Spain. Phone: 34 968 36 46 83. Fax: 34 968 36 41 50. E-mail: mtherrer{at}um.es

Published ahead of print on 6 August 2008.

P. R. Lowenstein and M. T. Herrero contributed equally to this work.

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Journal of Virology, October 2008, p. 9978-9993, Vol. 82, No. 20
0022-538X/08/$08.00+0     doi:10.1128/JVI.01326-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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