Characterization of CD8+ T Cells and Microenvironment in Oral Lesions of Human Immunodeficiency Virus-Infected Persons with Oropharyngeal Ca
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感染与免疫杂志 2005年第6期
Department of Oral Medicine
Department of General Dentistry
Department of Microbiology, Immunology, and Parasitology Dentistry and Biometerials
Center of Excellence in Oral and Craniofacial Biology, Louisiana State University School of Dentistry, 1100 Florida Avenue, New Orleans, Louisiana 70119
ABSTRACT
Oropharyngeal candidiasis (OPC), the most common oral infection in human immunodeficiency virus-positive persons, correlates with reduced blood CD4+ T cells. In those with OPC, CD8+ T cells accumulate at the lamina propria-epithelium interface at a distance from the organism at the outer epithelium. The present study aimed to characterize the tissue-associated CD8+ T cells and tissue microenvironment in both OPC+ and OPC– persons. The results show that the majority of CD8+ T cells possess the T-cell receptor, the thymus-derived CD8 antigen heterodimer, and similar levels of the 47, 41, and e7 homing receptors. Studies to evaluate the tissue microenvironment showed that in OPC+ persons, the adhesion molecule for T cells to enter mucosa, mucosal addressin cell adhesion molecule, is significantly increased, whereas E-cadherin, which allows T cells to migrate through mucosa, is significantly decreased compared to OPC– persons. These results continue to support a role for CD8+ T cells against OPC under conditions of reduced numbers of CD4+T cells, with susceptibility to infection potentially associated with a dysfunction in mucosal CD8+ T-cell migration by reduced tissue-associated E-cadherin.
INTRODUCTION
Oropharyngeal candidiasis (OPC), caused by Candida albicans, is the most common oral infection in those with human immunodeficiency virus (HIV) infection (18, 21, 23, 27). C. albicans is a ubiquitous fungal organism that is part of the normal microflora of the gastrointestinal and reproductive tracts. As a result of early exposure, most healthy individuals exhibit Candida-specific immunity that protects against infection. However, under immunocompromised conditions, such as HIV infection, C. albicans is capable of rapid conversion to a pathogen, causing symptomatic mucosal infections (8, 13, 21, 22, 27, 31). Clinically, OPC can be observed in lesions as a mixture of hyphae and yeast, normally located in the stratum corneum-keratin layer of the outer epithelium, and can affect the buccal mucosa, gingival cuff, palate, and tongue. The infections can be erythematous, atrophic lesions that appear reddish or pseudomembranous, white curd-like lesions, commonly referred to as thrush (10). OPC can lead to difficulty in chewing, painful swallowing, and ultimately reduced nutritional consumption with significant morbidity (17).
Cell-mediated immunity by CD4+ Th1-type cells is considered the predominant host defense mechanism against OPC (16, 18, 20, 21, 25, 31, 33, 34, 37). This is consistent with the frequency of OPC in HIV+ persons when blood CD4+ T-cell numbers drop below 200 cells/μl (18, 20-22, 25, 34, 39). Despite the strong correlation of increased incidence of OPC in people with reduced blood CD4+ T cells, immunological analyses have revealed little or no Candida-specific defects in blood CD4+ T cells (25). Thus, it is postulated that protection against OPC is multifactorial but primarily dependent on a threshold level of blood CD4+ T cells (usually 200 cells/μl). Below the CD4 cell threshold, systemic Th1-type cell-mediated immunity is no longer protective, and protection becomes dependent on several local immune mechanisms (14), including Th cytokines in saliva, epithelial cell anti-Candida activity, and the local presence of CD8+ T cells (26, 28, 43).
Although CD8+ T cells have not been considered prominent in host defense against Candida, putative roles for CD8+ T cells have been suggested from both animal models and clinical data (9, 28). In HIV+ persons, CD8+ T cells have been observed in OPC lesions, often exclusively (28). We recently reported that CD8+ T cells accumulate at the lamina propria-epithelium interface within OPC lesions at a considerable distance from the site of infection at the outer epithelium (28). This CD8+ T-cell accumulation was not observed in tissue from HIV– persons or HIV+ OPC– patients. Based on these results, it is postulated that CD8+ T cells do indeed play a role in host defense against OPC but that a dysfunction exists either in the cells or in the microenvironment, resulting in the inability of the cells to migrate through the mucosa.
T cells express specific homing receptors that interact with cell adhesion molecules (CAMs) in tissue that facilitate the migration of cells (reviewed in reference 19). These interactions, initiated by binding of CAMs to reciprocal homing receptors, play important roles in the mediation of the immune response against infections. For example, the binding of 47 on T cells with mucosal addressin CAM (MAdCAM) on tissue promotes the migration of those cells into mucosal tissue, while the interaction of e7 with E-cadherin on tissue facilitates the migration of T cells through mucosal tissues.
The purpose of this study was to explore potential cellular and/or microenvironmental dysfunctions by characterizing the CD8+ T cells, as well as the adhesion molecules on the oral tissue, during episodes of OPC.
MATERIALS AND METHODS
Study population. Patients were recruited and evaluated at the Louisiana State University Health Sciences Center HIV+ Outpatient Dental Clinic associated with the HIV Outpatient Program of the Medical Center of Louisiana at New Orleans and the Louisiana State University School of Dentistry. Informed consent was obtained from all participants and patients, and all procedures were followed in the conduct of clinical research in accordance with the Institutional Review Board at Louisiana State University Health Sciences Center. The subjects were part of a cohort (n = 473) established between 1998 and 2003 comprising 124 HIV-negative persons and 349 HIV-infected persons, including 128 HIV+ OPC+ and 221 HIV+ OPC– persons. A subset of the cohort, using banked specimens prospectively, was used for the present immunohistochemistry and RNA analyses, including 31 HIV+ OPC+ and 39 HIV+ OPC– persons, based on original power analyses and the general trends seen during the experimental procedures. Of these, 87% of OPC+ individuals had <200 blood CD4 cells/μl, whereas for OPC– individuals, 33% had <200 blood CD4 cells/μl and 77% had <500 blood CD4 cells/μl. In the OPC+ group, the average blood CD4 and CD8 cell counts were 115 and 445 cells/μl, respectively, and the average HIV load was 219,000 copies/ml. In the OPC– group, the average CD4 and CD8 cell counts were 321 and 862 cells/μl, respectively, and the average viral load was 159,000 copies/ml. Fifty-one percent of the HIV+ persons in the subset were receiving highly active antiretroviral therapy (HAART). In this cohort, HAART is defined as three or more antiretroviral medications with at least one being a protease inhibitor. Due to uncertainty in compliance, failures of HAART were not able to be effectively identified. Finally, 11 HIV– healthy volunteers were used as controls for specific assays.
Diagnosis of oropharyngeal candidiasis and detection of oral yeast colonization. The diagnosis of OPC and detection of oral yeast colonization were described previously (28, 42). Briefly, diagnosis of OPC was made based on the clinical appearance of oral mucosa, i.e., red, atrophic areas (erythematous) or white curd-like plaques (pseudomembranous). To confirm the presence of Candida in each biopsy specimen taken, the specific site was swabbed and cultured. Oral swabs were cultured on Sabouraud dextrose agar (Becton Dickinson Microbiology Systems, Franklin, NJ) and Chromagar (CHROMagar Microbiology, Paris, France). Identification of OPC was further confirmed by hyphae or blastoconidia present on a wet-mount slide preparation using potassium hydroxide (KOH), a positive swab culture with characteristic colony morphology, and a silver stain of the tissue section from the lesions, as previously described (28), to confirm the presence of the organism. Initial speciation was screened for by color on Chromagar. Green colonies were processed for germ tube formation, and nongreen colonies were identified to species level by API biochemical tests (API ID 32C; BioMerieux, Durham, N.C.). Only those patients with pseudomembranous OPC were included in the subcohort due to the extremely small numbers of erythematous OPC, as well as the differences in sites of infection that would not allow appropriate comparisons. Of the OPC+ patients in the subcohort (n = 31), lesions from all but 2 patients were identified as having C. albicans exclusively. Of the remaining two patients, one patient was infected with C. glabrata and the other with C. dubliniensis. In addition, four OPC+ patients had evidence of a mixed infection within the lesion in which C. albicans was found together with C. glabrata (n = 3) or C. krusei (n = 1). Of the 77% of OPC– patients asymptomatically colonized with yeast, 96% were colonized with C. albicans and 4% were colonized with non-albicans Candida species (C. glabrata or C. dubliniensis). These data were comparable to those of our previous study (28).
Specimen collection and processing. (i) Biopsy. The collection of buccal mucosa biopsy specimens was described previously (28). For immunohistochemistry, tissue sections (5 μm) were collected on glass slides, fixed in ice-cold acetone (5 min), and stored at –20°C. Total tissue RNA was extracted using Ultraspec RNA (Biotecx Laboratories, Inc., Houston, TX). RNA was quantified by Warburg-Christian equation and stored at –80°C.
(ii) PBLs. Venous blood (10 ml) was collected, and peripheral blood lymphocytes (PBLs) were isolated by differential centrifugation using Ficoll-Paque (Amersham Biosciences Corp., Piscataway, NJ). The PBLs were used for flow cytometry and RNA extraction.
Antibodies. The following antibodies were used for immunohistochemical staining: monoclonal mouse anti-human CD8 (DAKO Cytomation, Carpinteria, CA), T-cell receptor (TCR) (Pierce Biotechnology, Inc., Rockford, IL), 4 (CD49d) (BD Biosciences Pharmingen, San Diego, CA), e (CD103) (DAKO Cytomation), 1 (CD29) (BD Biosciences Pharmingen), ICAM (CD54) (BD Biosciences Pharmingen), MAdCAM-1 (Oncogene Research Products, San Diego, CA), and VCAM-1 (CD106) (EMD Biosciences, Inc., San Diego, CA) antibodies, with appropriate isotype control antibody (mouse immunoglobulin G1[IgG1]) (DAKO Cytomation); monoclonal mouse anti-human TCR antibody (Pierce Biotechnology, Inc.), and E-cadherin antibody (BD Biosciences Pharmingen) with appropriate isotype control antibody (mouse IgG2b) (Serotec, Inc., Raleigh, NC); monoclonal mouse anti-human CD8 antibody (Serotec, Inc.) with isotype control antibody (mouse IgG2a) (Serotec, Inc.); monoclonal rat anti-human 7 antibody (BD Biosciences Pharmingen) with isotype control antibody (rat IgG2a) (Serotec, Inc.); monoclonal rat anti-human CD8 antibody (Serotec, Inc.) with isotype control antibody (rat IgG2b) (Serotec, Inc.); fluorescein isothiocyanate (FITC)-conjugated monoclonal mouse anti-human CD3 antibody (BD Biosciences Pharmingen) with FITC-conjugated isotype control antibody (mouse IgG2a) (BD Biosciences Pharmingen); phycoerythrin (PE)-conjugated monoclonal mouse anti-human CD3 and CD8 ( subunit) antibodies (BD Biosciences Pharmingen) with PE-conjugated isotype control antibody (mouse IgG1) (BD Biosciences); monoclonal mouse anti-human CD8 antibody (Serotec, Inc.) with isotype control antibody (mouse IgG2a) (Serotec, Inc.) and FITC-conjugated rat anti-mouse IgG2a secondary antibody (BD Biosciences).
Immunohistochemistry. Immunohistochemical staining of buccal mucosa using chromogen and fluorescence has been previously described, as was hematoxylin and eosin staining (28).
(i) Chromogen staining. Briefly, all steps were performed at 4°C using the Anti-Mouse Cell and Tissue Staining Kit (horseradish peroxidase-3-amino-9-ethylcarbazole; R&D Systems, Minneapolis, MN). Serial sections were warmed for 5 min at room temperature; rehydrated in phosphate-buffered saline (PBS); blocked with 3% hydrogen peroxide, mouse serum, avidin, and biotin; and then incubated overnight with primary antibodies (1 μg/ml to 50 μg/ml). The treated slides were washed and incubated with appropriate anti-mouse (R&D Systems) or anti-rat (Vector Laboratories, Inc., Burlingame, CA) biotinylated IgG secondary antibody (5 μg/ml) for 1 h. The washed slides were then incubated for 30 min with high-sensitivity streptavidin-horseradish peroxidase conjugate (R&D Systems), washed, and incubated with the substrate 3-amino-9-ethylcarbazole chromogen (R&D Systems) for 10 min. Mayer's hematoxylin (Fisher Diagnostics, Fair Lawn, NJ) was used as a counterstain. The slides were preserved by using Crystal Mount aqueous mounting medium solution (Biomedia, Foster City, CA).
(ii) Fluorescence staining. Dual-color fluorescence staining was performed. All steps were performed at room temperature, except for the incubations with primary antibody, which were done overnight at 4°C. Serial sections were warmed to room temperature, treated for 10 min in ice-cold acetone, rinsed with PBS, blocked with blocking solution (2% normal serum, 1% bovine serum albumin, and 0.2% Triton X-100 in PBS), and incubated overnight with the first primary antibody (1 μg/ml to 10 μg/ml). The labeled slides were washed and incubated with appropriate biotinylated IgG secondary antibody (5 μg/ml; Vector Laboratories, Inc.) for 30 min. The washed slides were incubated with streptavidin-Alexa Fluor 594 (red) (20 μg/ml; Molecular Probes, Inc., Eugene, OR) for 30 min and washed. For the second label, the washed slides were incubated overnight with a second primary antibody (1 μg/ml to 10 μg/ml). Subsequent secondary-antibody labels were repeated as described above and stained with streptavidin-Alexa Fluor 488 (green) (20 μg/ml; Molecular Probes, Inc.) for 30 min. The washed slides were rinsed with water and allowed to dry at 4°C. The slides were preserved with Vectashield Hard Set mounting medium for fluorescence (Vector, Burlingame, CA), followed by coverslips.
RNase protection assay. Total RNA was analyzed using the Multi-Probe RNase Protection Assay System (BD Biosciences Pharmingen). Specific radiolabeled anti-sense RNA probes, human cell surface antigen (hCD-1; 50 ng), were hybridized to complementary mRNA in each sample of total buccal mucosa RNA (15 μg) and then digested with RNases A (0.048 ng) and T1 (1,500 U). The RNase-protected fragments were purified and resolved on a 5% Tris-borate-EDTA urea gel. The expressed mRNA species for each sample were identified by the presence of bands corresponding to the expected fragment lengths based on the undigested probe. Levels of specific mRNA were quantified using phosphorimaging technology (Amersham Biosciences Corp., Piscataway, NJ). Data were normalized to the densitometer value for CD45 (lymphocyte marker) and expressed as a ratio to CD45 mRNA.
Flow cytometry. Dual-color flow cytometry was performed with PBLs. Cells (1 x 106) were pelleted in Eppendorf tubes, blocked on ice with 2% bovine serum albumin for 20 min, and incubated with unconjugated, PE-conjugated, or FITC-conjugated primary antibodies (anti-CD3, -CD8, and -CD8; 10 μg/ml in PBS-2% fetal calf serum) for 30 min on ice. After the incubation time, the cells were washed twice with PBS-2% fetal calf serum, further incubated with conjugated secondary antibodies (0.5 μg/ml) when necessary, and washed as described above (15). Specific labeling was confirmed using appropriate isotype controls. Compensation for each fluorochrome was determined by parallel single-color analysis of cells labeled with one of each fluorochrome-conjugated antibody. Samples were analyzed by EPICS Elite flow cytometer (Beckman Coulter, Fullerton, CA) using EXPO 32 MultiCOMP software (Beckman Coulter).
Statistics. (i) Morphometric analysis. Using bright-field microscopy, 10 to 12 adjacent areas at a magnification of x4 or 2 to 4 adjacent areas at x10 (37,000 μm2/each) per slide were identified in areas of cell concentration (usually the lamina propria-epithelial border). These areas were gated, and positively stained T cells or stained areas were marked ("painted") using MetaView software (Universal Imaging Corp., Downington, PA). A percent threshold of positively stained cells per multiple units of area was quantified for each patient tested. These values were used to determine the mean and standard error of the mean for each patient group. The MIXED procedure in SAS 9v1 was used to analyze a mixed-effects model that considered OPC status (plus or minus) as a fixed effect and subject as a random effect. This model appropriately handles the repeated measurements obtained for each subject as clustered observations. Analysis was performed using a mixed-model analysis of variance test. Significance was defined as a P value of <0.05.
(ii) Densitometry analysis. Differences in mRNA values were identified using the unpaired Student's t test. Significant differences were defined as a P value of <0.05 using a two-tailed test. Statistics were performed using GraphPad Prism (GraphPad Software, San Diego, CA).
RESULTS
TCR expression on CD8+ T cells in oral tissue of OPC+ patients. To characterize the T cells present in OPC+ tissue, serial sectioned biopsy specimens of the buccal mucosa from OPC+ and OPC– patients were immunohistochemically labeled with anti- TCR or anti- TCR antibodies and detected by chromophore. Figure 1 shows representative results from both OPC+ and OPC– tissues. Isotype controls (not shown) showed no significant labeling. TCR+ (Fig. 1A) cells and TCR+ (Fig. 1B) cells were present in both lesion-positive and -negative tissues, with TCR+ cells predominating. Both cell types appeared to increase in OPC+ compared to OPC– tissues. However, morphometric quantitative analysis (Fig. 1C) showed significant increases only for TCR+ cells in OPC+ compared to OPC– tissue (P = 0.0214).
A second approach employed fluorescent labeling and confocal microscopy to evaluate the TCRs on CD8+ T cells. For this, tissue sections were dual labeled with anti-CD8 antibody conjugated to streptavidin-Alexa Fluor 594 (red) and either anti- TCR or anti- TCR antibodies conjugated to streptavidin-Alexa Fluor 488 (green). Figure 2 shows a representative result for OPC+ and OPC– tissue. The yellow cells in both uninfected (Fig. 2A and B) and infected (Fig. 2C and D) tissues show that the vast majority of TCR+ (Fig. 2A and C) and TCR+ (Fig. 2B and D) cells are CD8+. Figure 2E and F shows the chromogen staining for CD8+ cells in OPC– and OPC+ persons, respectively, providing a reference for the fluorescent images.
A third approach employed RNase protection assays on OPC– and OPC+ tissue mRNAs. Figure 3A shows a representative gel showing more abundant TCR mRNA than TCR mRNA, which is similar to PBLs. As expected, quantitative analysis showed an increase in TCR mRNA in OPC+ compared to OPC– tissue that approached statistical significance (P = 0.053), while no differences were observed in TCR mRNA (P = 0.941) (Fig. 3B).
CD8 chain expression in oral tissue. We next determined whether the CD8 antigen was composed of the normal heterodimer or the rarer homodimer. RNase protection assays (a representative image is shown in Fig. 4A) showed more CD8 than CD8 mRNA in OPC+ tissue, as well as in HIV-negative PBL mRNA. Tissue from OPC– persons was similar (data not shown). To further investigate this finding, suggestive of some CD8 + cells, oral tissue was dual labeled with anti-CD8 antibody conjugated to streptavidin-Alexa Fluor 594 and anti-CD8 antibody conjugated to streptavidin-Alexa Fluor 488. Representative results shown in Fig. 4B show that while most CD8+ T cells possess the and chains, a small number of cells appear to possess only chains. To examine this observation more closely, HIV-negative PBLs were labeled with anti-CD3 and anti-CD8 or -CD8 and evaluated by flow cytometry. Figure 4C illustrates that while all CD8+ cells were CD3+, only 75% of CD8+ cells were CD3+, revealing that 25% of CD8+ cells were non-T cells.
Integrin expression of T cells in OPC. To evaluate integrin expression on the CD8+ T cells in the oral lesions of those with OPC, OPC– and OPC+ tissues were immunohistochemically labeled with anti-4, -e, -1, and -7 antibodies both for single-chromophore detection and in various combinations for fluorescent confocal detection. Figure 5 shows representative results from serial buccal mucosa sections of OPC+ persons. Isotype controls (not shown) had no significant staining. Chromogen single labels (Fig. 5A) show that all four integrins are detectable. The general levels of expression were 4 > 1 > e > 7. Figure 5B shows various levels of integrin combinations. In this patient, the integrin expression showed 41 > e7 > 47. The prominent single-integrin staining patterns (green and red) in alternate combination staining preps (41 versus 47 or e7 versus 47) are consistent with the most colocalized integrins (41), confirming 41 as the most common integrin combination. The colocalization of 41 and 47 varied between patients with no consistent pattern for any group. However, the colocalization patterns were always consistent with whatever combination of integrins was most frequent for a potential patient. In contrast, e7 was present at similar levels in all patients. Results were similar in HIV+ OPC– persons and HIV– persons (data not shown).
Adhesion molecule expression in oral tissue. In addition to integrin expression, we also evaluated the reciprocal cell adhesion molecules (ICAM, MAdCAM, and E-cadherin) on the same OPC+ and OPC– buccal mucosal tissues. Representative images are shown in Fig. 6. ICAM (Fig. 6A and D) was found in both the lamina propria and epithelium with similar levels observed in OPC– (Fig. 6A) and OPC+ (Fig. 6D) tissue, as well as tissue from HIV– persons (data not shown). MAdCAM and E-cadherin expression were restricted predominately to the epithelium. MAdCAM (Fig. 6B and E) expression was generally increased while E-cadherin (Fig. 6C and F) expression was visibly decreased in OPC+ (Fig. 6E and F) compared to OPC– (Fig. 6B and C) tissues, including those for HIV– persons (data not shown). Quantitative analysis showed virtually equivalent ICAM expression in OPC+ versus OPC– tissue (Fig. 7A). In contrast, MAdCAM expression in OPC+ tissues was significantly increased (Fig. 7B) (P = 0.03). Results did not differ for ICAM or MAdCAM if OPC+ tissue was limited to those with visibly accumulated CD8+ T cells (data not shown). In the case of E-cadherin expression compared to OPC– tissues, a significant reduction was found in OPC+ tissue when limited to those with cellular accumulation (Fig. 7C) (>50%) (P = 0.005), but not with all tissues inclusively (P > 0.05) (data not shown).
DISCUSSION
The accumulation of CD8+ T cells in OPC+ lesions suggests a role for CD8+ T cells against OPC. However, inasmuch as the presence of the cells suggested some role, the distance of the cells from Candida at the outer epithelium indicated a potential dysfunction in either the cells or the microenvironment that inhibited the migration of cells to the organism.
The present study further characterized the CD8+ T cells and the microenvironment relative to cellular migration. The results showed that a vast majority of the T cells in the tissue expressed TCR, although there were considerable numbers of TCR+ cells. On average, 80 to 85% of the cells expressed TCR. The 15 to 20% TCR+ cell proportion, while a minor T-cell population, is higher than in blood (2 to 3%). This higher level of TCR+ cells is similar to what is observed in the vagina (15, 30), as well as skin and the gastrointestinal tract (4, 5, 24, 32). In OPC+ persons, the cellular increase at the epithelium-lamina propria interface was largely composed of TCR+ cells, as the TCR+ cells were only slightly elevated and loosely distributed within the tissue. Fluorescent confocal microscopy showed that the TCR+ cells present were CD8+, consistent with the presence of CD8+ lymphocytes in previous reports of cellular evaluation in OPC (29, 36, 46). Thus, TCR+ CD8+ T cells appear to be a major lymphocyte population present in OPC lesions. Indirect evidence for responses by TCR+ cells in the oral tissue comes from several animal models of OPC that show a role for CD4 (2, 3, 11, 12, 40) and/or CD8+ T cells against OPC (2, 3, 9). The only exception was in an immunocompetent animal model of OPC, where although both and TCR+ cells were observed in the oral cavity, TCR+ cells correlated with clearance of the experimental infection (1, 7).
The CD8 antigen on the cells in the tissue was found to consist of the common heterodimer, similar to CD8+ cells in the peripheral circulation, with no evidence for the alternative extrathymically derived homodimer (24, 35). This was not initially definite, as considerably more chain was detected in tissue by both RNase protection assays and confocal immunohistochemical staining. However, flow cytometry of PBLs from HIV-negative persons revealed that a small population of non-T cells expressed the CD8 chain. The identity of these CD8a+ CD3– cells is unclear, but one possibility is a subset of natural killer (NK) cells that have been identified as CD2+ CD3– CD8+ or CD2– CD3– CD8+ in blood (41). Thus, it is suspected that a similar non-T-cell CD8 chain also accounts for the higher chain presence in the oral tissue. This is even more plausible if one assumes the CD8+ cells in the tissue are derived from the peripheral circulation. In any case, results were similar for OPC– and OPC+ tissues, suggesting that the CD8 antigen on cells migrating to, or proliferating in, the oral mucosa was the thymically derived heterodimer.
Recruitment of leukocytes in the tissue is a multistep process involving the interaction between integrins and their protein ligands. Integrin expression on the cells in oral biopsy tissue showed the presence of all four integrins evaluated (4, e, 1, and 7) that combine to form homing receptors for migration into and through mucosal tissue. Interestingly, confocal microscopy showed various levels of combinations of integrins, with some patients having more 41 than 47 and vice versa. e7, however, was more consistent, but was usually expressed at lower levels than 41 or 47. (Fluorescent dual-label staining confirmed that the homing receptors were present on CD8+ T cells [data not shown].) There was no difference in the general distribution of homing receptors between OPC+ and OPC– tissues, although direct comparisons were difficult due to the various combinations of integrins per patient. Nevertheless, except for the obvious increase in homing-receptor-positive cells in OPC+ tissue as a result of increased CD8+ cells, there did not appear to be any evidence for a difference in homing receptor expression on the cells present in those with OPC.
In contrast to the homing receptors, evaluation of reciprocal adhesion molecule expression showed considerable differences between OPC– and OPC+ tissues. While ICAM levels were similar in both tissues, consistent with its constitutive production (6, 38, 45), MAdCAM was significantly increased in OPC+ persons. This clearly supports the presence of CD8+ T cells in the tissue and suggests that the cells did indeed migrate from the peripheral circulation rather than proliferate from within the tissue. Most notable, however, was the significant reduction in E-cadherin in OPC+ tissue with accumulated CD8+ T cells. This represents a possible dysfunction in the local microenvironment of this subset of OPC+ patients. As E-cadherin is the molecule responsible for the migration of T cells through mucosal tissue, together with the consistent presence of the reciprocal homing receptor e7 on the CD8+ T cells in the tissue, a reduction in E-cadherin may account for the accumulation of the CD8+ T cells at the lamina propria-epithelium interface in these OPC+ persons. Of particular interest is the predominant expression of E-cadherin, or the lack thereof, in the epithelium rather than the lamina propria, which further supports this concept. Obviously, however, this applies only to the subset of OPC+ patients with accumulated CD8+ T cells, although they are the majority of OPC+ cases. It is unclear what may be associated with the susceptibility to infection in those with increased, but not visibly accumulated, CD8+ T cells. In such cases, the CD8+ T cells and/or other unidentified cofactors may be involved. An alternative explanation is that the accumulated CD8+ T cells are a normal response to infection and effectively form a barrier against dissemination by Candida. In that case, reduced E-cadherin, which also functions to form tight junctions between epithelial cells (44), may enhance adherence and invasion by Candida where gaps occur in the epithelium, thus promoting an infectious state. This is unlikely, however, when those patients with OPC who do not have reduced E-cadherin or any evidence of accumulated CD8+ T cells are taken into account.
Together, the results from the majority of patients support our overall hypothesis of a role for CD8+ T cells in host defense against OPC but a dysfunction in the microenvironment of those with OPC. We postulate that the high constitutive E-cadherin expression in the epithelia of OPC– persons allows the migration of CD4+ or CD8+ T cells to the outer epithelium when necessary and of CD8+ T cells exclusively when CD4+ T cells are below a protection threshold. However, in a subset of patients, a reduction in E-cadherin expression in the epithelium prohibits the migration of CD8+ T cells, resulting in accumulation at the epithelium-lamina propria interface. To our knowledge, this is the first observation of its kind revealing a potential mechanism for susceptibility to infection involving the adhesion molecules and the microenvironment rather than the effector cells. Nevertheless, this certainly supports the site-specific nature of infection by this commensal organism in the HIV-infected population. In fact, this finding is quite distinct from the results for vaginal candidiasis, where a similar lack of involvement by T cells occurs because of reduced infiltration into the mucosa by T cells with reduced homing receptors, whereas adhesion molecule expression is normal (45).
While the putative CD8+ effector cells appear normal in those with OPC, studies to evaluate other factors of immune status (i.e., costimulation, chemokine receptors, and functional activity) will be necessary to confirm this and potentially to identify other factors of susceptibility in those without accumulated CD8+ T cells. Of equal importance are studies to more fully understand the reduced E-cadherin expression on the tissues of those with OPC and accumulated CD8+ T cells. For this, a longitudinal study will be necessary to determine whether the reduced expression is transient or permanent, and if transient, under what conditions. We have evaluated some cofactors in E-cadherin dynamics (viral load, sexual behavior, intravenous drug use, and HAART). Preliminary results from this small cohort show no correlation of reduced E-cadherin with any of these possible cofactors. However, this will require confirmation by a larger cohort. Interestingly, although the HIV load does not appear to play a prominent role, that does not preclude an effect of HIV on the oral-tissue microenvironment. Indeed, OPC is more common in HIV+ persons than in those with other types of immunosuppression (transplantation or treatment for lymphoma).
In summary, we have extended our observations of the immune events taking place in those persons with OPC. The latest results continue to support the hypothesis that CD8+ T cells play some role in host defense against OPC. However, instead of susceptibility to OPC being manifested by a putative dysfunction in the CD8+ T cells, it appears that at least in a majority of patients, a putative dysfunction occurs in the microenvironment whereby a reduction in E-cadherin may be associated with susceptibility to infection.
ACKNOWLEDGMENTS
This work was supported by a National Institutes of Health Public Health Service Grant (DE-12178) from the National Institute of Dental and Craniofacial Research.
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Department of General Dentistry
Department of Microbiology, Immunology, and Parasitology Dentistry and Biometerials
Center of Excellence in Oral and Craniofacial Biology, Louisiana State University School of Dentistry, 1100 Florida Avenue, New Orleans, Louisiana 70119
ABSTRACT
Oropharyngeal candidiasis (OPC), the most common oral infection in human immunodeficiency virus-positive persons, correlates with reduced blood CD4+ T cells. In those with OPC, CD8+ T cells accumulate at the lamina propria-epithelium interface at a distance from the organism at the outer epithelium. The present study aimed to characterize the tissue-associated CD8+ T cells and tissue microenvironment in both OPC+ and OPC– persons. The results show that the majority of CD8+ T cells possess the T-cell receptor, the thymus-derived CD8 antigen heterodimer, and similar levels of the 47, 41, and e7 homing receptors. Studies to evaluate the tissue microenvironment showed that in OPC+ persons, the adhesion molecule for T cells to enter mucosa, mucosal addressin cell adhesion molecule, is significantly increased, whereas E-cadherin, which allows T cells to migrate through mucosa, is significantly decreased compared to OPC– persons. These results continue to support a role for CD8+ T cells against OPC under conditions of reduced numbers of CD4+T cells, with susceptibility to infection potentially associated with a dysfunction in mucosal CD8+ T-cell migration by reduced tissue-associated E-cadherin.
INTRODUCTION
Oropharyngeal candidiasis (OPC), caused by Candida albicans, is the most common oral infection in those with human immunodeficiency virus (HIV) infection (18, 21, 23, 27). C. albicans is a ubiquitous fungal organism that is part of the normal microflora of the gastrointestinal and reproductive tracts. As a result of early exposure, most healthy individuals exhibit Candida-specific immunity that protects against infection. However, under immunocompromised conditions, such as HIV infection, C. albicans is capable of rapid conversion to a pathogen, causing symptomatic mucosal infections (8, 13, 21, 22, 27, 31). Clinically, OPC can be observed in lesions as a mixture of hyphae and yeast, normally located in the stratum corneum-keratin layer of the outer epithelium, and can affect the buccal mucosa, gingival cuff, palate, and tongue. The infections can be erythematous, atrophic lesions that appear reddish or pseudomembranous, white curd-like lesions, commonly referred to as thrush (10). OPC can lead to difficulty in chewing, painful swallowing, and ultimately reduced nutritional consumption with significant morbidity (17).
Cell-mediated immunity by CD4+ Th1-type cells is considered the predominant host defense mechanism against OPC (16, 18, 20, 21, 25, 31, 33, 34, 37). This is consistent with the frequency of OPC in HIV+ persons when blood CD4+ T-cell numbers drop below 200 cells/μl (18, 20-22, 25, 34, 39). Despite the strong correlation of increased incidence of OPC in people with reduced blood CD4+ T cells, immunological analyses have revealed little or no Candida-specific defects in blood CD4+ T cells (25). Thus, it is postulated that protection against OPC is multifactorial but primarily dependent on a threshold level of blood CD4+ T cells (usually 200 cells/μl). Below the CD4 cell threshold, systemic Th1-type cell-mediated immunity is no longer protective, and protection becomes dependent on several local immune mechanisms (14), including Th cytokines in saliva, epithelial cell anti-Candida activity, and the local presence of CD8+ T cells (26, 28, 43).
Although CD8+ T cells have not been considered prominent in host defense against Candida, putative roles for CD8+ T cells have been suggested from both animal models and clinical data (9, 28). In HIV+ persons, CD8+ T cells have been observed in OPC lesions, often exclusively (28). We recently reported that CD8+ T cells accumulate at the lamina propria-epithelium interface within OPC lesions at a considerable distance from the site of infection at the outer epithelium (28). This CD8+ T-cell accumulation was not observed in tissue from HIV– persons or HIV+ OPC– patients. Based on these results, it is postulated that CD8+ T cells do indeed play a role in host defense against OPC but that a dysfunction exists either in the cells or in the microenvironment, resulting in the inability of the cells to migrate through the mucosa.
T cells express specific homing receptors that interact with cell adhesion molecules (CAMs) in tissue that facilitate the migration of cells (reviewed in reference 19). These interactions, initiated by binding of CAMs to reciprocal homing receptors, play important roles in the mediation of the immune response against infections. For example, the binding of 47 on T cells with mucosal addressin CAM (MAdCAM) on tissue promotes the migration of those cells into mucosal tissue, while the interaction of e7 with E-cadherin on tissue facilitates the migration of T cells through mucosal tissues.
The purpose of this study was to explore potential cellular and/or microenvironmental dysfunctions by characterizing the CD8+ T cells, as well as the adhesion molecules on the oral tissue, during episodes of OPC.
MATERIALS AND METHODS
Study population. Patients were recruited and evaluated at the Louisiana State University Health Sciences Center HIV+ Outpatient Dental Clinic associated with the HIV Outpatient Program of the Medical Center of Louisiana at New Orleans and the Louisiana State University School of Dentistry. Informed consent was obtained from all participants and patients, and all procedures were followed in the conduct of clinical research in accordance with the Institutional Review Board at Louisiana State University Health Sciences Center. The subjects were part of a cohort (n = 473) established between 1998 and 2003 comprising 124 HIV-negative persons and 349 HIV-infected persons, including 128 HIV+ OPC+ and 221 HIV+ OPC– persons. A subset of the cohort, using banked specimens prospectively, was used for the present immunohistochemistry and RNA analyses, including 31 HIV+ OPC+ and 39 HIV+ OPC– persons, based on original power analyses and the general trends seen during the experimental procedures. Of these, 87% of OPC+ individuals had <200 blood CD4 cells/μl, whereas for OPC– individuals, 33% had <200 blood CD4 cells/μl and 77% had <500 blood CD4 cells/μl. In the OPC+ group, the average blood CD4 and CD8 cell counts were 115 and 445 cells/μl, respectively, and the average HIV load was 219,000 copies/ml. In the OPC– group, the average CD4 and CD8 cell counts were 321 and 862 cells/μl, respectively, and the average viral load was 159,000 copies/ml. Fifty-one percent of the HIV+ persons in the subset were receiving highly active antiretroviral therapy (HAART). In this cohort, HAART is defined as three or more antiretroviral medications with at least one being a protease inhibitor. Due to uncertainty in compliance, failures of HAART were not able to be effectively identified. Finally, 11 HIV– healthy volunteers were used as controls for specific assays.
Diagnosis of oropharyngeal candidiasis and detection of oral yeast colonization. The diagnosis of OPC and detection of oral yeast colonization were described previously (28, 42). Briefly, diagnosis of OPC was made based on the clinical appearance of oral mucosa, i.e., red, atrophic areas (erythematous) or white curd-like plaques (pseudomembranous). To confirm the presence of Candida in each biopsy specimen taken, the specific site was swabbed and cultured. Oral swabs were cultured on Sabouraud dextrose agar (Becton Dickinson Microbiology Systems, Franklin, NJ) and Chromagar (CHROMagar Microbiology, Paris, France). Identification of OPC was further confirmed by hyphae or blastoconidia present on a wet-mount slide preparation using potassium hydroxide (KOH), a positive swab culture with characteristic colony morphology, and a silver stain of the tissue section from the lesions, as previously described (28), to confirm the presence of the organism. Initial speciation was screened for by color on Chromagar. Green colonies were processed for germ tube formation, and nongreen colonies were identified to species level by API biochemical tests (API ID 32C; BioMerieux, Durham, N.C.). Only those patients with pseudomembranous OPC were included in the subcohort due to the extremely small numbers of erythematous OPC, as well as the differences in sites of infection that would not allow appropriate comparisons. Of the OPC+ patients in the subcohort (n = 31), lesions from all but 2 patients were identified as having C. albicans exclusively. Of the remaining two patients, one patient was infected with C. glabrata and the other with C. dubliniensis. In addition, four OPC+ patients had evidence of a mixed infection within the lesion in which C. albicans was found together with C. glabrata (n = 3) or C. krusei (n = 1). Of the 77% of OPC– patients asymptomatically colonized with yeast, 96% were colonized with C. albicans and 4% were colonized with non-albicans Candida species (C. glabrata or C. dubliniensis). These data were comparable to those of our previous study (28).
Specimen collection and processing. (i) Biopsy. The collection of buccal mucosa biopsy specimens was described previously (28). For immunohistochemistry, tissue sections (5 μm) were collected on glass slides, fixed in ice-cold acetone (5 min), and stored at –20°C. Total tissue RNA was extracted using Ultraspec RNA (Biotecx Laboratories, Inc., Houston, TX). RNA was quantified by Warburg-Christian equation and stored at –80°C.
(ii) PBLs. Venous blood (10 ml) was collected, and peripheral blood lymphocytes (PBLs) were isolated by differential centrifugation using Ficoll-Paque (Amersham Biosciences Corp., Piscataway, NJ). The PBLs were used for flow cytometry and RNA extraction.
Antibodies. The following antibodies were used for immunohistochemical staining: monoclonal mouse anti-human CD8 (DAKO Cytomation, Carpinteria, CA), T-cell receptor (TCR) (Pierce Biotechnology, Inc., Rockford, IL), 4 (CD49d) (BD Biosciences Pharmingen, San Diego, CA), e (CD103) (DAKO Cytomation), 1 (CD29) (BD Biosciences Pharmingen), ICAM (CD54) (BD Biosciences Pharmingen), MAdCAM-1 (Oncogene Research Products, San Diego, CA), and VCAM-1 (CD106) (EMD Biosciences, Inc., San Diego, CA) antibodies, with appropriate isotype control antibody (mouse immunoglobulin G1[IgG1]) (DAKO Cytomation); monoclonal mouse anti-human TCR antibody (Pierce Biotechnology, Inc.), and E-cadherin antibody (BD Biosciences Pharmingen) with appropriate isotype control antibody (mouse IgG2b) (Serotec, Inc., Raleigh, NC); monoclonal mouse anti-human CD8 antibody (Serotec, Inc.) with isotype control antibody (mouse IgG2a) (Serotec, Inc.); monoclonal rat anti-human 7 antibody (BD Biosciences Pharmingen) with isotype control antibody (rat IgG2a) (Serotec, Inc.); monoclonal rat anti-human CD8 antibody (Serotec, Inc.) with isotype control antibody (rat IgG2b) (Serotec, Inc.); fluorescein isothiocyanate (FITC)-conjugated monoclonal mouse anti-human CD3 antibody (BD Biosciences Pharmingen) with FITC-conjugated isotype control antibody (mouse IgG2a) (BD Biosciences Pharmingen); phycoerythrin (PE)-conjugated monoclonal mouse anti-human CD3 and CD8 ( subunit) antibodies (BD Biosciences Pharmingen) with PE-conjugated isotype control antibody (mouse IgG1) (BD Biosciences); monoclonal mouse anti-human CD8 antibody (Serotec, Inc.) with isotype control antibody (mouse IgG2a) (Serotec, Inc.) and FITC-conjugated rat anti-mouse IgG2a secondary antibody (BD Biosciences).
Immunohistochemistry. Immunohistochemical staining of buccal mucosa using chromogen and fluorescence has been previously described, as was hematoxylin and eosin staining (28).
(i) Chromogen staining. Briefly, all steps were performed at 4°C using the Anti-Mouse Cell and Tissue Staining Kit (horseradish peroxidase-3-amino-9-ethylcarbazole; R&D Systems, Minneapolis, MN). Serial sections were warmed for 5 min at room temperature; rehydrated in phosphate-buffered saline (PBS); blocked with 3% hydrogen peroxide, mouse serum, avidin, and biotin; and then incubated overnight with primary antibodies (1 μg/ml to 50 μg/ml). The treated slides were washed and incubated with appropriate anti-mouse (R&D Systems) or anti-rat (Vector Laboratories, Inc., Burlingame, CA) biotinylated IgG secondary antibody (5 μg/ml) for 1 h. The washed slides were then incubated for 30 min with high-sensitivity streptavidin-horseradish peroxidase conjugate (R&D Systems), washed, and incubated with the substrate 3-amino-9-ethylcarbazole chromogen (R&D Systems) for 10 min. Mayer's hematoxylin (Fisher Diagnostics, Fair Lawn, NJ) was used as a counterstain. The slides were preserved by using Crystal Mount aqueous mounting medium solution (Biomedia, Foster City, CA).
(ii) Fluorescence staining. Dual-color fluorescence staining was performed. All steps were performed at room temperature, except for the incubations with primary antibody, which were done overnight at 4°C. Serial sections were warmed to room temperature, treated for 10 min in ice-cold acetone, rinsed with PBS, blocked with blocking solution (2% normal serum, 1% bovine serum albumin, and 0.2% Triton X-100 in PBS), and incubated overnight with the first primary antibody (1 μg/ml to 10 μg/ml). The labeled slides were washed and incubated with appropriate biotinylated IgG secondary antibody (5 μg/ml; Vector Laboratories, Inc.) for 30 min. The washed slides were incubated with streptavidin-Alexa Fluor 594 (red) (20 μg/ml; Molecular Probes, Inc., Eugene, OR) for 30 min and washed. For the second label, the washed slides were incubated overnight with a second primary antibody (1 μg/ml to 10 μg/ml). Subsequent secondary-antibody labels were repeated as described above and stained with streptavidin-Alexa Fluor 488 (green) (20 μg/ml; Molecular Probes, Inc.) for 30 min. The washed slides were rinsed with water and allowed to dry at 4°C. The slides were preserved with Vectashield Hard Set mounting medium for fluorescence (Vector, Burlingame, CA), followed by coverslips.
RNase protection assay. Total RNA was analyzed using the Multi-Probe RNase Protection Assay System (BD Biosciences Pharmingen). Specific radiolabeled anti-sense RNA probes, human cell surface antigen (hCD-1; 50 ng), were hybridized to complementary mRNA in each sample of total buccal mucosa RNA (15 μg) and then digested with RNases A (0.048 ng) and T1 (1,500 U). The RNase-protected fragments were purified and resolved on a 5% Tris-borate-EDTA urea gel. The expressed mRNA species for each sample were identified by the presence of bands corresponding to the expected fragment lengths based on the undigested probe. Levels of specific mRNA were quantified using phosphorimaging technology (Amersham Biosciences Corp., Piscataway, NJ). Data were normalized to the densitometer value for CD45 (lymphocyte marker) and expressed as a ratio to CD45 mRNA.
Flow cytometry. Dual-color flow cytometry was performed with PBLs. Cells (1 x 106) were pelleted in Eppendorf tubes, blocked on ice with 2% bovine serum albumin for 20 min, and incubated with unconjugated, PE-conjugated, or FITC-conjugated primary antibodies (anti-CD3, -CD8, and -CD8; 10 μg/ml in PBS-2% fetal calf serum) for 30 min on ice. After the incubation time, the cells were washed twice with PBS-2% fetal calf serum, further incubated with conjugated secondary antibodies (0.5 μg/ml) when necessary, and washed as described above (15). Specific labeling was confirmed using appropriate isotype controls. Compensation for each fluorochrome was determined by parallel single-color analysis of cells labeled with one of each fluorochrome-conjugated antibody. Samples were analyzed by EPICS Elite flow cytometer (Beckman Coulter, Fullerton, CA) using EXPO 32 MultiCOMP software (Beckman Coulter).
Statistics. (i) Morphometric analysis. Using bright-field microscopy, 10 to 12 adjacent areas at a magnification of x4 or 2 to 4 adjacent areas at x10 (37,000 μm2/each) per slide were identified in areas of cell concentration (usually the lamina propria-epithelial border). These areas were gated, and positively stained T cells or stained areas were marked ("painted") using MetaView software (Universal Imaging Corp., Downington, PA). A percent threshold of positively stained cells per multiple units of area was quantified for each patient tested. These values were used to determine the mean and standard error of the mean for each patient group. The MIXED procedure in SAS 9v1 was used to analyze a mixed-effects model that considered OPC status (plus or minus) as a fixed effect and subject as a random effect. This model appropriately handles the repeated measurements obtained for each subject as clustered observations. Analysis was performed using a mixed-model analysis of variance test. Significance was defined as a P value of <0.05.
(ii) Densitometry analysis. Differences in mRNA values were identified using the unpaired Student's t test. Significant differences were defined as a P value of <0.05 using a two-tailed test. Statistics were performed using GraphPad Prism (GraphPad Software, San Diego, CA).
RESULTS
TCR expression on CD8+ T cells in oral tissue of OPC+ patients. To characterize the T cells present in OPC+ tissue, serial sectioned biopsy specimens of the buccal mucosa from OPC+ and OPC– patients were immunohistochemically labeled with anti- TCR or anti- TCR antibodies and detected by chromophore. Figure 1 shows representative results from both OPC+ and OPC– tissues. Isotype controls (not shown) showed no significant labeling. TCR+ (Fig. 1A) cells and TCR+ (Fig. 1B) cells were present in both lesion-positive and -negative tissues, with TCR+ cells predominating. Both cell types appeared to increase in OPC+ compared to OPC– tissues. However, morphometric quantitative analysis (Fig. 1C) showed significant increases only for TCR+ cells in OPC+ compared to OPC– tissue (P = 0.0214).
A second approach employed fluorescent labeling and confocal microscopy to evaluate the TCRs on CD8+ T cells. For this, tissue sections were dual labeled with anti-CD8 antibody conjugated to streptavidin-Alexa Fluor 594 (red) and either anti- TCR or anti- TCR antibodies conjugated to streptavidin-Alexa Fluor 488 (green). Figure 2 shows a representative result for OPC+ and OPC– tissue. The yellow cells in both uninfected (Fig. 2A and B) and infected (Fig. 2C and D) tissues show that the vast majority of TCR+ (Fig. 2A and C) and TCR+ (Fig. 2B and D) cells are CD8+. Figure 2E and F shows the chromogen staining for CD8+ cells in OPC– and OPC+ persons, respectively, providing a reference for the fluorescent images.
A third approach employed RNase protection assays on OPC– and OPC+ tissue mRNAs. Figure 3A shows a representative gel showing more abundant TCR mRNA than TCR mRNA, which is similar to PBLs. As expected, quantitative analysis showed an increase in TCR mRNA in OPC+ compared to OPC– tissue that approached statistical significance (P = 0.053), while no differences were observed in TCR mRNA (P = 0.941) (Fig. 3B).
CD8 chain expression in oral tissue. We next determined whether the CD8 antigen was composed of the normal heterodimer or the rarer homodimer. RNase protection assays (a representative image is shown in Fig. 4A) showed more CD8 than CD8 mRNA in OPC+ tissue, as well as in HIV-negative PBL mRNA. Tissue from OPC– persons was similar (data not shown). To further investigate this finding, suggestive of some CD8 + cells, oral tissue was dual labeled with anti-CD8 antibody conjugated to streptavidin-Alexa Fluor 594 and anti-CD8 antibody conjugated to streptavidin-Alexa Fluor 488. Representative results shown in Fig. 4B show that while most CD8+ T cells possess the and chains, a small number of cells appear to possess only chains. To examine this observation more closely, HIV-negative PBLs were labeled with anti-CD3 and anti-CD8 or -CD8 and evaluated by flow cytometry. Figure 4C illustrates that while all CD8+ cells were CD3+, only 75% of CD8+ cells were CD3+, revealing that 25% of CD8+ cells were non-T cells.
Integrin expression of T cells in OPC. To evaluate integrin expression on the CD8+ T cells in the oral lesions of those with OPC, OPC– and OPC+ tissues were immunohistochemically labeled with anti-4, -e, -1, and -7 antibodies both for single-chromophore detection and in various combinations for fluorescent confocal detection. Figure 5 shows representative results from serial buccal mucosa sections of OPC+ persons. Isotype controls (not shown) had no significant staining. Chromogen single labels (Fig. 5A) show that all four integrins are detectable. The general levels of expression were 4 > 1 > e > 7. Figure 5B shows various levels of integrin combinations. In this patient, the integrin expression showed 41 > e7 > 47. The prominent single-integrin staining patterns (green and red) in alternate combination staining preps (41 versus 47 or e7 versus 47) are consistent with the most colocalized integrins (41), confirming 41 as the most common integrin combination. The colocalization of 41 and 47 varied between patients with no consistent pattern for any group. However, the colocalization patterns were always consistent with whatever combination of integrins was most frequent for a potential patient. In contrast, e7 was present at similar levels in all patients. Results were similar in HIV+ OPC– persons and HIV– persons (data not shown).
Adhesion molecule expression in oral tissue. In addition to integrin expression, we also evaluated the reciprocal cell adhesion molecules (ICAM, MAdCAM, and E-cadherin) on the same OPC+ and OPC– buccal mucosal tissues. Representative images are shown in Fig. 6. ICAM (Fig. 6A and D) was found in both the lamina propria and epithelium with similar levels observed in OPC– (Fig. 6A) and OPC+ (Fig. 6D) tissue, as well as tissue from HIV– persons (data not shown). MAdCAM and E-cadherin expression were restricted predominately to the epithelium. MAdCAM (Fig. 6B and E) expression was generally increased while E-cadherin (Fig. 6C and F) expression was visibly decreased in OPC+ (Fig. 6E and F) compared to OPC– (Fig. 6B and C) tissues, including those for HIV– persons (data not shown). Quantitative analysis showed virtually equivalent ICAM expression in OPC+ versus OPC– tissue (Fig. 7A). In contrast, MAdCAM expression in OPC+ tissues was significantly increased (Fig. 7B) (P = 0.03). Results did not differ for ICAM or MAdCAM if OPC+ tissue was limited to those with visibly accumulated CD8+ T cells (data not shown). In the case of E-cadherin expression compared to OPC– tissues, a significant reduction was found in OPC+ tissue when limited to those with cellular accumulation (Fig. 7C) (>50%) (P = 0.005), but not with all tissues inclusively (P > 0.05) (data not shown).
DISCUSSION
The accumulation of CD8+ T cells in OPC+ lesions suggests a role for CD8+ T cells against OPC. However, inasmuch as the presence of the cells suggested some role, the distance of the cells from Candida at the outer epithelium indicated a potential dysfunction in either the cells or the microenvironment that inhibited the migration of cells to the organism.
The present study further characterized the CD8+ T cells and the microenvironment relative to cellular migration. The results showed that a vast majority of the T cells in the tissue expressed TCR, although there were considerable numbers of TCR+ cells. On average, 80 to 85% of the cells expressed TCR. The 15 to 20% TCR+ cell proportion, while a minor T-cell population, is higher than in blood (2 to 3%). This higher level of TCR+ cells is similar to what is observed in the vagina (15, 30), as well as skin and the gastrointestinal tract (4, 5, 24, 32). In OPC+ persons, the cellular increase at the epithelium-lamina propria interface was largely composed of TCR+ cells, as the TCR+ cells were only slightly elevated and loosely distributed within the tissue. Fluorescent confocal microscopy showed that the TCR+ cells present were CD8+, consistent with the presence of CD8+ lymphocytes in previous reports of cellular evaluation in OPC (29, 36, 46). Thus, TCR+ CD8+ T cells appear to be a major lymphocyte population present in OPC lesions. Indirect evidence for responses by TCR+ cells in the oral tissue comes from several animal models of OPC that show a role for CD4 (2, 3, 11, 12, 40) and/or CD8+ T cells against OPC (2, 3, 9). The only exception was in an immunocompetent animal model of OPC, where although both and TCR+ cells were observed in the oral cavity, TCR+ cells correlated with clearance of the experimental infection (1, 7).
The CD8 antigen on the cells in the tissue was found to consist of the common heterodimer, similar to CD8+ cells in the peripheral circulation, with no evidence for the alternative extrathymically derived homodimer (24, 35). This was not initially definite, as considerably more chain was detected in tissue by both RNase protection assays and confocal immunohistochemical staining. However, flow cytometry of PBLs from HIV-negative persons revealed that a small population of non-T cells expressed the CD8 chain. The identity of these CD8a+ CD3– cells is unclear, but one possibility is a subset of natural killer (NK) cells that have been identified as CD2+ CD3– CD8+ or CD2– CD3– CD8+ in blood (41). Thus, it is suspected that a similar non-T-cell CD8 chain also accounts for the higher chain presence in the oral tissue. This is even more plausible if one assumes the CD8+ cells in the tissue are derived from the peripheral circulation. In any case, results were similar for OPC– and OPC+ tissues, suggesting that the CD8 antigen on cells migrating to, or proliferating in, the oral mucosa was the thymically derived heterodimer.
Recruitment of leukocytes in the tissue is a multistep process involving the interaction between integrins and their protein ligands. Integrin expression on the cells in oral biopsy tissue showed the presence of all four integrins evaluated (4, e, 1, and 7) that combine to form homing receptors for migration into and through mucosal tissue. Interestingly, confocal microscopy showed various levels of combinations of integrins, with some patients having more 41 than 47 and vice versa. e7, however, was more consistent, but was usually expressed at lower levels than 41 or 47. (Fluorescent dual-label staining confirmed that the homing receptors were present on CD8+ T cells [data not shown].) There was no difference in the general distribution of homing receptors between OPC+ and OPC– tissues, although direct comparisons were difficult due to the various combinations of integrins per patient. Nevertheless, except for the obvious increase in homing-receptor-positive cells in OPC+ tissue as a result of increased CD8+ cells, there did not appear to be any evidence for a difference in homing receptor expression on the cells present in those with OPC.
In contrast to the homing receptors, evaluation of reciprocal adhesion molecule expression showed considerable differences between OPC– and OPC+ tissues. While ICAM levels were similar in both tissues, consistent with its constitutive production (6, 38, 45), MAdCAM was significantly increased in OPC+ persons. This clearly supports the presence of CD8+ T cells in the tissue and suggests that the cells did indeed migrate from the peripheral circulation rather than proliferate from within the tissue. Most notable, however, was the significant reduction in E-cadherin in OPC+ tissue with accumulated CD8+ T cells. This represents a possible dysfunction in the local microenvironment of this subset of OPC+ patients. As E-cadherin is the molecule responsible for the migration of T cells through mucosal tissue, together with the consistent presence of the reciprocal homing receptor e7 on the CD8+ T cells in the tissue, a reduction in E-cadherin may account for the accumulation of the CD8+ T cells at the lamina propria-epithelium interface in these OPC+ persons. Of particular interest is the predominant expression of E-cadherin, or the lack thereof, in the epithelium rather than the lamina propria, which further supports this concept. Obviously, however, this applies only to the subset of OPC+ patients with accumulated CD8+ T cells, although they are the majority of OPC+ cases. It is unclear what may be associated with the susceptibility to infection in those with increased, but not visibly accumulated, CD8+ T cells. In such cases, the CD8+ T cells and/or other unidentified cofactors may be involved. An alternative explanation is that the accumulated CD8+ T cells are a normal response to infection and effectively form a barrier against dissemination by Candida. In that case, reduced E-cadherin, which also functions to form tight junctions between epithelial cells (44), may enhance adherence and invasion by Candida where gaps occur in the epithelium, thus promoting an infectious state. This is unlikely, however, when those patients with OPC who do not have reduced E-cadherin or any evidence of accumulated CD8+ T cells are taken into account.
Together, the results from the majority of patients support our overall hypothesis of a role for CD8+ T cells in host defense against OPC but a dysfunction in the microenvironment of those with OPC. We postulate that the high constitutive E-cadherin expression in the epithelia of OPC– persons allows the migration of CD4+ or CD8+ T cells to the outer epithelium when necessary and of CD8+ T cells exclusively when CD4+ T cells are below a protection threshold. However, in a subset of patients, a reduction in E-cadherin expression in the epithelium prohibits the migration of CD8+ T cells, resulting in accumulation at the epithelium-lamina propria interface. To our knowledge, this is the first observation of its kind revealing a potential mechanism for susceptibility to infection involving the adhesion molecules and the microenvironment rather than the effector cells. Nevertheless, this certainly supports the site-specific nature of infection by this commensal organism in the HIV-infected population. In fact, this finding is quite distinct from the results for vaginal candidiasis, where a similar lack of involvement by T cells occurs because of reduced infiltration into the mucosa by T cells with reduced homing receptors, whereas adhesion molecule expression is normal (45).
While the putative CD8+ effector cells appear normal in those with OPC, studies to evaluate other factors of immune status (i.e., costimulation, chemokine receptors, and functional activity) will be necessary to confirm this and potentially to identify other factors of susceptibility in those without accumulated CD8+ T cells. Of equal importance are studies to more fully understand the reduced E-cadherin expression on the tissues of those with OPC and accumulated CD8+ T cells. For this, a longitudinal study will be necessary to determine whether the reduced expression is transient or permanent, and if transient, under what conditions. We have evaluated some cofactors in E-cadherin dynamics (viral load, sexual behavior, intravenous drug use, and HAART). Preliminary results from this small cohort show no correlation of reduced E-cadherin with any of these possible cofactors. However, this will require confirmation by a larger cohort. Interestingly, although the HIV load does not appear to play a prominent role, that does not preclude an effect of HIV on the oral-tissue microenvironment. Indeed, OPC is more common in HIV+ persons than in those with other types of immunosuppression (transplantation or treatment for lymphoma).
In summary, we have extended our observations of the immune events taking place in those persons with OPC. The latest results continue to support the hypothesis that CD8+ T cells play some role in host defense against OPC. However, instead of susceptibility to OPC being manifested by a putative dysfunction in the CD8+ T cells, it appears that at least in a majority of patients, a putative dysfunction occurs in the microenvironment whereby a reduction in E-cadherin may be associated with susceptibility to infection.
ACKNOWLEDGMENTS
This work was supported by a National Institutes of Health Public Health Service Grant (DE-12178) from the National Institute of Dental and Craniofacial Research.
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