αEβ7 (CD103) Expression Identifies a Highly Active, Tonsil-Resident Effector-Memory CTL Population

Tonia Woodberry, Todd J. Suscovich, Leah M. Henry, Meredith August, Michael T. Waring, Amitinder Kaur, Christoph Hess, Jeffery L. Kutok, Jon C. Aster, Frederick Wang, David T. Scadden and Christian Brander
J Immunol October 1, 2005, 175 (7) 4355-4362; DOI: https://doi.org/10.4049/jimmunol.175.7.4355

Abstract

The characterization of antiviral CTL responses has largely been limited to assessing Ag-specific immune responses in the peripheral blood. Consequently, there is an incomplete understanding of the cellular immune responses at mucosal sites where many viruses enter and initially replicate and how the Ag specificity and activation status of CTL derived from these mucosal sites may differ from that of blood-derived CTL. In this study, we show that EBV-specific CTL responses in the tonsils are of comparable specificity and breadth but of a significantly higher magnitude compared with responses in the peripheral blood. EBV-specific, tonsil-resident, but not PBMC-derived, T cells expressed the integrin/activation marker CD103 (αEβ7), consistent with the detection of its ligand, E-cadherin, on tonsillar squamous cells. These CD8-positive, CD103-positive, tonsil-derived CTL were largely CCR7- and CD45RA- negative effector-memory cells and responded to lower Ag concentrations in in vitro assays than their CD103-negative PBMC-derived counterparts. Thus, EBV-specific CTL in the tonsil, a crucial site for EBV entry and replication, are of greater magnitude and phenotypically distinct from CTL in the peripheral blood and may be important for effective control of this orally transmitted virus.

Epstein-Barr virus is one of the most prevalent human viral infections, and the oral cavity has been identified as the site of initial EBV infection as well as a reservoir for lifelong viral persistence (1, 2). Yet, relatively little is known about virus-specific cellular immune responses at the site of viral entry. In particular, the nature of antiviral lymphocytes in the tonsil, their induction upon infection, and Ag specificity compared with lymphocytes in the peripheral blood is poorly understood. In the present study, we compared EBV- and HIV-specific T cell responses in the tonsil and the peripheral blood to better understand if, and how, virus-specific immune responses at the site of viral entry and replication differ from those detected in the peripheral blood.

EBV- and HIV-specific CD4 and CD8 T cell responses in the peripheral blood have been well characterized during both acute and chronic viral infection (3, 4, 5, 6, 7, 8). Data from a number of studies indicate that T cells from MALT can significantly differ in their maturation status and phenotypes compared with peripheral blood-derived T cells (9, 10, 11, 12, 13, 14, 15, 16, 17). Particularly, HIV- and CMV-specific tonsil- and blood-derived T cells have been shown to differ in their perforin production, CD28 expression, and breadth of virus-specific CTL responses (17). A more recent, and more extensive, study has also documented that although TCR repertoires of EBV-specific CTL in the tonsil and PBMC were largely overlapping, tonsil-derived cells expressed significantly more CD28 (15). Together, these studies suggest that the local tonsil environment imparts functional and phenotypic differences on tonsil-resident CTL, rather than merely reflecting the presence of different T cell populations in various body compartments (15). Thus, potential functional and phenotypic differences between peripheral and mucosal T cells may be directly associated with the ability of lymphocytes to circulate from the peripheral blood to mucosal sites, including the intestinal mucosa and the tonsils.

Mucosal environments have indeed been shown to harbor lymphocytes that express a number of specific adhesion molecules, including the intestinal homing receptors α4β7 and the integrin and activation marker αEβ7 (CD103) (18, 19, 20, 21, 22, 23). In particular, α4β7 has been shown to bind to the mucosal addressin mucosal addressin cell adhesion molecule-1, which is selectively expressed on the capillary endothelium in the gastrointestinal tract, whereas CD103 has been shown to bind E-cadherin, an integrin receptor known to be expressed on intestinal endothelial cells, thymic tissue, and hepatocytes (21, 24, 25, 26). Furthermore, CD103 has been described as an intestinal intraepithelial lymphocyte and activation marker (21, 22, 27). Thus, it is plausible that CD103 expression not only allows homing or retention of T cells in these sites, but may also be associated with an increased T cell activation status. Although no study has demonstrated E-cadherin expression in the tonsil, the finding that sputum-derived T lymphocytes and leukocytes in bronchoalveolar lavages of healthy adults express significant amounts of CD103 suggests that E-cadherin and CD103 could be important for the retention of tonsil-resident CTL (22, 28, 29, 30).

Since the tonsil is a major reservoir for EBV, we assessed whether EBV-specific cells isolated from tonsillar tissue would differ in their phenotype, magnitude, and specificity compared with cells isolated from the peripheral blood. Specifically, we asked whether tonsillar T lymphocytes would express the integrin CD103 and whether they would reveal a more activated effector phenotype than their counterparts in the peripheral blood. This enrichment in EBV- but not HIV-specific CTL in the tonsil, along with the expression of the CD103 ligand, E-cadherin, on squamous cells of the tonsillar epithelium provide a link between a viral reservoir and the expansion of a site-specific immunity that may be important for the control of viral replication and dissemination.

Materials and Methods

Study subjects and oral tissue samples

The characteristics of study subjects are summarized in Tables I⇓ and II⇓. Tonsil biopsies and peripheral blood samples were obtained from a total of 11 individuals, all with chronic EBV infection, as determined by the presence of detectable EBV-specific IgG (5). Three of these subjects were also HIV coinfected and did (L859, L8154) or did not (K47) receive antiretroviral therapy. Oral samples were obtained from tonsillar biopsies in eight individuals and from a sublingual biopsy in one subject. The tonsillar biopsies yielded 6–100 million mononuclear cells, with no difference in the yield from the HIV-negative or HIV-positive samples (median of 18 and 16 million, respectively). Two additional individuals donated parts of their tonsils after a tonsillectomy performed to relieve sleep apnea. No inflammation or other signs of tonsillitis were observed in any of the individuals. The whole tonsils yielded 600 million and 1.1 billion mononuclear cells. The study was approved by the Institutional Review Boards of Massachusetts General Hospital, and each subject provided written informed consent before enrollment. For all subjects, peripheral blood samples were obtained immediately before the biopsies were performed. PBMC were isolated by density gradient centrifugation (Histopaque 1077; Sigma-Aldrich) within 24 h of venipuncture. Tonsil tissue was immediately placed on ice and processed within 2 h of surgical removal by mechanical disruption using scissors and passage over a 100-μm cell strainer (BD Labware). Isolated tonsillar cells and PBMC were washed and tested directly without prior in vitro expansion. High-resolution HLA typing was performed at the HLA typing laboratory at Massachusetts General Hospital using sequence-specific primer PCR as previously described (31).

View this table:
Table I.

Cohort information

View this table:
Table II.

CD103 expression on T cells in tonsil tissue and PBMC

Peptides

EBV-derived CTL epitopes used to screen for EBV-specific CTL responses have been previously described (5, 32). Optimal HIV class I-restricted CTL epitopes were used to screen for HIV-specific cellular responses in the three HIV-coinfected subjects (33). Only described epitopes known to be presented by the individual’s HLA class I type were used. Peptides were synthesized at the peptide synthesis facility at Massachusetts General Hospital using F-moc chemistry.

Flow cytometry

Tonsillar cells and PBMC were stained with CD3, CD4, CD8, and CD19 surface Abs (BD Biosciences) and analyzed by flow cytometry to determine the T and B cell ratios in each sample and to normalize the magnitude of responses to the same input CD8 T cell number as indicated. Phenotypic analyses were performed by staining cells with αEβ7 (Immunotech)-, α4β7 (BD Biosciences)-, CD69 (BD Biosciences)-, CXCR3 (BD Biosciences)-, CCR7 (R&D Systems)-, CD45RA (BD Biosciences)-, or CD45RO (BD Biosciences)-specific Abs as described elsewhere (34).

Tetramer binding

A total of 1 × 106 tonsillar cells or PBMC was incubated at 37°C for 30 min with tetramers and subsequently stained with surface Abs at room temperature as described previously (35). Three different EBV-specific tetramers were used, presenting either the HLA-B8-restricted lytic epitope RAKFKQLL (B8-RAK) derived from EBV-BZLF1 protein, the epitope FLRGRAYGL (B8-FLR) derived from EBNA-3A, or the HLA-A2-restricted lytic epitope GLCTLVAML (A2-GLC) derived from EBV-BMLF1 (5). Epitope-containing tetramers were prepared as described elsewhere (36).

ELISPOT assay

Freshly isolated tonsillar cells and PBMC were tested directly in in vitro ELISPOT assays as previously described (3). One hundred thousand to 200,000 PBMC and 100,000–300,000 tonsil cells were added to each well in 100 μl of RPMI 1640 supplemented with 10% FCS (Sigma-Aldrich). Peptides were added at a final concentration of 10 μg/ml. No peptide was added to three to five wells, which served as negative controls. PHA (Remel) was added at a concentration of 1.8 μg/ml as a positive control. After overnight incubation at 37°C, plates were developed and the number of spots was determined using the AID ELISPOT Reader Unit (Autoimmun Diagnostika). Results were expressed as spot-forming cells per 106 input CD8 T cells to compensate for variable CD8 T cell proportions in the tonsil cells and the PBMC. Control experiments in which tonsil cells were supplemented with peripheral blood cells or potent APC, such as EBV-transformed B cells lines and PHA blasts did not show increased response rates of tonsil cells, suggesting that APC function in the tonsil cell suspension was not impaired. The cutoff for positive responses was determined as a minimum of five spots per well or responses exceeding the mean of negative wells plus three times the SD, whichever gave the higher value.

Detection of E-cadherin in tonsil tissues

Immunohistochemistry was performed using 5-μm-thick Formalin-fixed, paraffin-embedded tissue sections. Briefly, slides were soaked in xylene, passed through graded alcohols, and put in distilled water. Slides were then pretreated with 10 mM citrate, pH 6.0 (Zymed Laboratories) in a steam pressure cooker (Decloaking Chamber; BioCare Medical) according to the manufacturer’s instructions, followed by washing in distilled water. All further steps were performed at room temperature in a hydrated chamber. Slides were pretreated with Peroxidase Block (DakoCytomation) for 5 min to quench endogenous peroxidase activity. Primary monoclonal anti-E-cadherin Ab (DakoCytomation) was applied at a 1/400 dilution in DakoCytomation diluent for 1 h. Slides were washed in 50 mM Tris-Cl (pH 7.4) and anti-mouse HRP-conjugated Ab (Envision detection kit; DakoCytomation) was applied for 30 min. After further washing, immunoperoxidase staining was developed using a diaminobenzidine chromogen kit (DakoCytomation) per the manufacturer’s protocol and counterstained with hematoxylin.

Statistical analysis

Results are presented as median values unless otherwise noted. Statistical analyses included the matched-pairs test (two tailed) for comparison of the breadth, magnitude, and phenotype of immune responses detected in the tonsil and the PBMC samples.

Results

Increased magnitude of EBV latent Ag-specific T cells in tonsils compared with PBMC

To assess virus-specific immune responses specific for an orally transmitted virus (EBV) and a virus not typically transmitted orally (HIV), tonsillar and peripheral blood samples were obtained from a total of 11 EBV-infected subjects, 3 of whom were HIV-1 coinfected (Table I⇑). The number of targeted CTL epitopes (breadth of response) and the total magnitude of responses detected (total spot-forming cells/106 CD8 T cells) in the tonsil vs the peripheral blood were assessed and compared in a direct ex vivo ELISPOT assay using previously described, optimally defined EBV or HIV CTL epitopes (32). Epitopes were selected based on the individual’s HLA class I type, and between 7 and 32 different epitopes were tested for each subject (5, 32, 37). The analyses revealed a comparable breadth of EBV-specific responses in tonsillar and peripheral T cells (Fig. 1⇓A). Although a trend toward a greater breadth of responses against latently expressed viral Ags in tonsillar T cells compared with peripheral T cells was observed (p = 0.052), the number of lytic epitopes targeted in tonsillar and peripheral T cells did not differ, as both compartments yielded a median of two detected responses (data not shown).

FIGURE 1.
FIGURE 1.

Stronger EBV-specific responses are detected in tonsil cells compared with PBMC. The breadth and magnitude of epitope-specific CTL responses was determined in blood (▴, ▵) and tonsil (•, ○) samples obtained either from HIV-negative (▪, ▴, •) or HIV-infected subjects (□, ▵, ○). A, The total breadth of responses (number of epitopes targeted per individual) is compared between the two compartments. B. The total magnitude of responses (sum of all individual responses) is compared for each subject between blood and tonsil. C, The total magnitude of responses against epitopes encoded by latent (▴ and ▵ and • and ○) or lytic (▪ and □ and ♦ and ⋄) EBV Ags in blood (▴, ▵) and tonsil (•, ○) is compared for each subject. All responses are normalized to spot-forming cells per 106 input CD8 T cells.

In contrast, when the magnitude of the total EBV epitope-specific response was compared, significantly stronger responses were detected in the tonsil compared with the blood (Fig. 1⇑B; p = 0.0064). This increase in magnitude was attributable to significantly stronger responses to latent but not to lytic epitopes in the tonsil (Fig. 1⇑C; p = 0.0026). Although the difference in total magnitude could have been due to the slightly increased median number of responses detected in the tonsil, a comparison of the magnitude of each individual response that was detectable in both compartments showed a significantly increased magnitude in the tonsil compared with the periphery (data not shown; p < 0.0001). These data were confirmed by intracellular cytokine analyses performed in six individuals, again showing stronger latent responses in tonsillar T cells compared with peripheral T cells (data not shown). Together, the data demonstrate that the tonsils harbored stronger EBV-specific T cell responses than the blood, with particularly strong tonsil-derived responses against epitopes derived from viral proteins expressed during EBV latency.

Similar breadth and magnitude of HIV-specific CTL responses in tonsillar cells and peripheral T cells

In contrast to EBV, the oral cavity is unlikely to serve as a major reservoir for HIV and therefore HIV-specific CTL responses in the tonsil may be of similar or reduced breadth and magnitude when compared with the blood. To test this, cells from tonsil biopsies obtained from three HIV-coinfected individuals were tested for HIV epitope-specific immune responses and compared with responses in the peripheral blood. Among the 3 subjects, responses to 12 HIV-derived CTL epitopes were tested in both compartments (3, 4, and 5 epitopes per subject, respectively). A total of 11 positive responses were detected in PBMC, with 9 of them also inducing positive responses in tonsillar tissue. Although there was only a trend toward stronger HIV-specific responses in the blood compared with the tonsils, EBV-specific responses were significantly stronger in the tonsil compared with the blood for these same three subjects (data not shown; p = 0.008). Thus, HIV responses appeared equivalent in terms of their breadth and magnitude in the tonsil compared with the blood, whereas responses to the orally transmitted EBV were increased in the tonsils of the same individuals.

Tonsil-resident T cells express an effector-memory phenotype and high levels of CD103

The increased frequency of EBV-specific T cells in the tonsillar tissue may be due to the continual stimulation or immigration of EBV-specific but not HIV-specific T cells in this compartment. To test whether the presence of EBV-specific T cells in the tonsil was indeed associated with the expression of specific adhesion or activation markers, tonsil- and blood-derived T cells were analyzed for the expression of adhesion/homing markers, including CXCR3, α4β7 and αEβ7 (CD103), and markers discriminating effector and naive T cells from effector-memory and central-memory T cells (22, 38, 39, 40, 41, 42).

Phenotyping showed a 2- to 10-fold higher percentage of CD8 T cells in the blood compared with the tonsils (p = 0.001), whereas CD4 T cells were enriched in tonsils relative to PBMC in 8 of the 10 individuals (Table II⇑). The CD4 and CD8 T cells from either compartment were tested for the expression of α4β7, a mucosal homing marker, and the chemokine receptor CXCR3, another molecule involved in cell trafficking and expressed after recent Ag contact (38, 43). The analyses revealed differences in the expression of both markers, as CD8 T cells in the tonsil expressed more frequently CXCR3 compared with the blood (Fig. 2⇓A; p = 0.034), whereas fewer tonsillar CD4 T cells expressed this homing marker (p = 0.027). In contrast, α4β7 expression was increased on blood CD4 T cells compared with tonsillar CD4 cells (p < 0.0001), yet its expression did not differ between CD8 T cells in the two compartments (Fig. 2⇓B). Tonsil-resident T cells were also found to express αEβ7 (CD103), an intraepithelial lymphocyte marker expressed on mucosa-associated T lymphocytes as well as on activated T cells and recent thymic emigrants (21, 25, 26, 44). On average, tonsil cells contained 16 times more CD103+CD8+ T cells and 3 times more CD103+CD4+ T cells compared with blood-derived CD4 and CD8 T cells (Fig. 2⇓C and Table II⇑; p < 0.0001 for CD8). These data show that CD103 expression was significantly elevated on tonsillar CD8 T cells compared with PBMC, while α4β7 staining was comparable between tonsil and blood-derived CD8 T cells.

FIGURE 2.
FIGURE 2.

Differential integrin/homing marker expression on T cells in tonsils and blood: CXCR3 (A), α4β7 (B), and CD103 (C) expression was determined on CD8 (▴, •) and CD4 (▵, ○) T cells derived from blood (▴, ▵) and tonsil (•, ○). The percentage of cells expressing the respective markers is indicated. Values of p are indicated only for comparisons where statistically significant differences were observed.

The maturation and activation phenotype of T cells in the tonsil and blood was further assessed using the previously described maturation and activation markers CCR7 and the CD45RA/RO isoforms (40, 41, 42). Total lymphocytes, as well as EBV-specific CTL, in tonsil samples or PBMC preparations, were stained with CCR7- and CD45RA-specific Abs. Total lymphocyte staining was performed on nine subjects, whereas for six subjects EBV-epitope tetramers were available to assess CCR7/CD45 expression on epitope-specific T cells in either compartment. A similar percentage of naive (CCR7+CD45RA+) and central memory cells (CCR7+CD45RA−) were found in both the Ag-specific and nonspecific lymphocytes derived from the tonsil and the blood (data not shown). In contrast, there were significant differences in the percentages of effector-memory (CCR7−CD45RA−) and effector (CCR7−CD45RA+) cells in both the total lymphocytes, as well as the Ag-specific CD8 T cells between the two compartments. In both cases, the tonsils expressed a higher percentage of effector-memory cells than the blood (total lymphocytes, p = 0.017 and Ag specific, p = 0.089). Conversely, effector cells were more frequently detected in the blood than in the tonsils (total lymphocytes, p = 0.016 and Ag specific, p = 0.043). Together, these data indicate that the CD8 T cell population in the tonsil is enriched for cells expressing an effector-memory phenotype both on total CD8 T cells and EBV Ag-specific CTL.

To show that epitope-specific cells with an effector-memory phenotype also expressed CD103, tetramer analyses using EBV latent and lytic Ag epitopes were combined with CD103 and CD45RO staining (Fig. 3⇓). The analyses included two individuals tested against lytic protein-derived CTL epitopes and one individual tested for a latent Ag-specific response. Regardless of the origin of the viral epitope, more tetramer-positive cells in the tonsil were CD103+ and CCR7−/CD45RO+ compared with the peripheral blood. In all three cases, the Ag-specific, tetramer-positive cells were at least 2-fold enriched in the tonsil compared with the peripheral blood. In addition, CD103+ effector-memory cell populations were at least 15 times more prevalent in the tonsils compared with those in blood. Similarly, CD103+CD45RO+ cells from the tonsil also more frequently expressed CD69 (65%) compared with CD8+CD103− tonsillar T cells (26%; data not shown). Together, the phenotypic analyses demonstrate that tonsil cells are enriched for EBV-specific CTL and that these cells express specific markers of an activated, effector-memory phenotype.

FIGURE 3.
FIGURE 3.

Epitope-specific effector-memory cells from tonsil but not blood express CD103: the expression of CD103 on CD8-positive, tetramer-positive T cells was determined in the tonsil and blood by flow cytometry. Cells were stained with tetramers (A2-GLC (EBV BMLF-1 protein, GLCTLVAML), B8-RAK (EBV BZLF-1 protein, RAKFKQLL), and B8-FLR (EBV EBNA-3A protein, FLRGRAYG), as well as Abs against CD103 and CD45RO, to indirectly assess the fraction of CD45RA-expressing, CD103+ effector-memory cells.

CD103-expressing, tonsil-derived CTL are highly reactive to low Ag concentration

Since CD103 has been described not only as an integrin but also as an activation marker (45), we assessed whether the significant increase in CD103+ cells in the tonsil would be reflected by these cells responding to decreasing Ag concentration. Total lymphocytes from the blood and the tonsil from two different individuals were tested by ELISPOT assay using decreasing amounts of peptide. As shown in Fig. 4⇓, detectable responses were observed at 10- to 100-fold lower concentrations of peptide in cells isolated from the tonsil when compared with those from blood. These data were confirmed in a third individual from whom tonsillar CD103+ and CD103− CD8 T cells were sorted by FACS and showed equal magnitude of responses using 100 times lower peptide concentrations in CD103+ cells compared with CD103− cells (Fig. 4⇓C). Together, the data indicate that CD103 expression is associated with a more activated, effector-memory-like phenotype in the tonsil, likely allowing these cells to respond to lower Ag concentrations than CD103− cells.

FIGURE 4.
FIGURE 4.

Tonsillar T cells respond to lower Ag concentrations more effectively than peripheral T cells. Tonsil cells (–•–) and PBMC (- - -▴- - -) from two patients were analyzed in duplicate in an ELISPOT assay using serial peptide dilutions of the BMRF-1-derived, HLA-A2-restricted epitope YVLDHLIVV (A) or the EBNA-3c-derived, HLA-B27-restricted epitope RRIYDLIEL (B). C, Tonsil cells from subject K18 were separated by FACS into CD103-positive and CD103-negative CD8 T cells. Forty thousand cells per well were plated in an ELISPOT assay and the LMP2-derived A2-CLG peptide was added at concentrations from 10 μg/μl to 10 pg/μl. The graph shows the number of spots detected at the indicated peptide concentrations. Insufficient CD8-positive CD103-positive tonsil T cells were obtained to test the 1-ng/μl peptide concentration. All responses are normalized to spot-forming cells per 106 input CD8 T cells.

The CD103 ligand E-cadherin is expressed in tonsils

CD103 has been identified on T cells derived from a number of tissues, including the liver (24), renal and pancreatic allografts (46, 47), thymus (44, 48), and rectal and vaginal mucosa (12, 49). Although alternative ligands for CD103 have been described (50), the infiltration or, alternatively, retention of CD103-expressing cells in the tonsil could be facilitated by the expression of E-cadherin in this tissue. Indeed, histological analyses showed tonsillar squamous epithelial cells strongly stained for E-cadherin, with no staining observed in infiltrating lymphoid cells (Fig. 5⇓). This is the first description of E-cadherin expression on tonsillar epithelial cells and is consistent with the possibility that E-cadherin contributes to the relative enrichment of CD103-expressing, EBV-specific CTL in the tonsils.

FIGURE 5.
FIGURE 5.

E-cadherin expression in tonsil. Tonsils stained for E-cadherin (diaminobenzidine chromogen, hematoxylin counterstain; original magnification, ×200) show strongly positive squamous epithelium (A) compared with negative staining control (B). Note that infiltrating lymphoid cells failed to stain for E-cadherin.

Discussion

The understanding of antiviral immune responses at mucosal sites in the body is incomplete, and it is currently unclear whether the peripheral blood and the oral mucosa are discrete immunological compartments or whether there is immunological bidirectional “spillover” between both sites. The present study aimed to identify differences in the magnitude, the specificity, and the activation status of antiviral CTL present between these sites and to associate these differences with the presence or absence of viral replication in the different compartments. Surprisingly, initial analyses revealed EBV-specific CTL responses of greater magnitude in the tonsil compared with the peripheral blood, which was largely mediated by increased CTL responses to epitopes derived from latently expressed viral proteins. This increase of EBV-specific CTL was observed even though the overall number of CD8 T cells in the tonsil was significantly reduced compared with those of the peripheral blood. However, this reduction did not compromise the breadth of the EBV responses (Fig. 1⇑).

The selective elevation of latent EBV-specific CTL responses in the tonsil may reflect the persistent presence of B cells latently infected with EBV. Although ongoing lytic Ag expression in the oral cavity has been described, it may be comparatively less frequent and possibly insufficient to maintain large lytic protein-specific CTL populations (51). Alternatively, latent EBV Ag-specific CTL responses in the peripheral blood may be selectively reduced compared with lytic responses, potentially due to reduced availability of latent Ag, since peripheral infected B cells express fewer EBV latent genes compared with infected tonsillar B cells (52). Clearly, additional studies in subjects with documented oral viral shedding at the time of tonsil biopsy or tonsillectomy are needed to provide further insight into the relationship between Ag expression and the magnitude of EBV-specific cellular immunity in the oral cavity. Furthermore, future studies would ideally include a more comprehensive testing of the total EBV-specific immunity. Because the 90 EBV-derived CTL epitopes described to date (32) are only derived from 12 different viral proteins, responses to other regions may be missed. Whether this would affect the responses detected in the tonsil, the blood, or both compartments will require a complete knowledge of CTL epitopes, which may be gained by screening tonsil in addition to PBMC against viral Ags.

Phenotypic analyses revealed significant differences between the CD8 T cell populations in tonsils and blood. In particular, a significantly higher proportion of the total and EBV-specific CD8 T cell population in the tonsil expressed the E-cadherin-binding integrin αEβ7 (CD103) when compared with blood-derived CD8 T cells (Fig. 2⇑). Our demonstration of E-cadherin expression in endothelial cells in the tonsil suggests that it serves as the receptor for CD103 on tonsil-resident cells (Fig. 5⇑). However, CD103 also binds the lymphocyte endothelial-epithelial cell adhesion molecule for which expression throughout tonsillar epithelia as well as on high endothelial venules in the tonsil has been demonstrated (50, 53). Thus, lymphoid cells in the crypt regions of tonsils, closely associated with the surface epithelium lymphocytes that express CD103, may bind either receptor, facilitating the immunosurveillance of nonintestinal epithelia (54). In support of this, CD103 expression has also been documented on T cells in the lacrimal and salivary glands of subjects with autoimmune diseases affecting the oral cavity such as Sjögren’s syndrome (55). In addition, some reports have found CD103 expression on what were considered regulatory T cells, and a murine study suggests an important role of CD103 in retaining these T cells at the site of Leishmania infection (56, 57). However, in addition to CD103, other surface markers known to play a role in lymphocyte homing and T cell retention may also be important in populating the tonsil tissue with virus-specific T cells. One possible candidate is CCR7, which is more frequently expressed on CTL directed against EBV latent Ags compared with CTL targeting lytic Ags, and which thus could, at least in part, mediate the increased latent Ag-specific CTL activity in the tonsil (16).

EBV-specific CD8 T cells and total CD8 T cells from tonsils also expressed low levels of CD45RA and CCR7, consistent with an effector-memory phenotype (40, 41, 42). In addition, these largely CD103+, effector-memory tonsil CD8 T cells were more reactive to their cognate Ag than the CD103− CD8 T cells (Fig. 4⇑), since they required ∼100 times lower Ag concentrations to respond to Ag in vitro. These findings are in line with the previously reported role of CD103 as a marker of T cell activation and suggests that retention of these highly sensitive cells in the tonsil may permit rapid recall responses at even low Ag concentrations. Sample availability from oral biopsies did not permit for the determination of EBV Ag, and larger numbers of full tonsil donations would be needed to test for a potential association between CTL responses viral gene expression in a statistically satisfactory manner. Nevertheless, the present data suggest a relationship between the anatomic location of viral infection and the quality of the specific CTL response, providing a mechanism by which an orally transmitted virus may induce different profiles of reactivity among lymphocytes in local vs systemic compartments. If this relationship holds for other viruses, it would have significant implications for vaccine development and highlight the importance of a detailed knowledge of cellular immune responses at specific sites of pathogen exposure, rather than the limited focus on the peripheral blood.

Acknowledgments

We thank Suqin He for generation of EBV tetramers.

Disclosures

The authors have no financial conflict of interest.

Footnotes

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

  • ↵1 This study was supported by National Institutes of Health/National Institute for Dental and Craniofacial Research Grant PO1 DE01438-01.

  • ↵2 T.W. and T.J.S. contributed equally to this work.

  • ↵3 Address correspondence and reprint requests to Dr. Christian Brander, Partners AIDS Research Center, Fifth Floor, MGH East, No. 5214, 149 13th Street, Charlestown, MA 02129. E-mail address: cbrander{at}partners.org

  • Received May 18, 2005.
  • Accepted July 28, 2005.

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