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The Journal of Immunology, 2006, 177: 479-491.
Copyright © 2006 by The American Association of Immunologists

Altered CD4+ T Cell Phenotype and Function Determine the Susceptibility to Mucosal Candidiasis in Transgenic Mice Expressing HIV-11

Daniel Lewandowski*, Miriam Marquis*, Francine Aumont*, Annie-Claude Lussier-Morin*, Marianne Raymond*, Serge Sénéchal*, Zaher Hanna{dagger},{ddagger},§, Paul Jolicoeur*,{ddagger},§ and Louis de Repentigny2,*,¶

* Department of Microbiology and Immunology, {dagger} Department of Medicine, Faculty of Medicine, University of Montreal, Montreal, Quebec, Canada; {ddagger} Laboratory of Molecular Biology, Clinical Research Institute of Montreal, Montreal, Quebec, Canada; § Division of Experimental Medicine, McGill University, Montreal, Quebec, Canada; and Sainte-Justine Hospital, Montreal, Quebec, Canada


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The impairments of protective mucosal immunity which cause susceptibility to oropharyngeal candidiasis (OPC) in HIV infection remain undefined. This study used a model of OPC in CD4C/HIV MutA transgenic (Tg) mice expressing Rev, Env, and Nef of HIV-1 to investigate the role of transgene expressing dendritic cells (DCs) and CD4+ T cells in maintenance of chronic oral carriage of Candida albicans. DCs were depleted in the Tg mice and had an immature phenotype, with low expression of MHC class II and IL-12. CD4+ T cells were quantitatively reduced in the oral mucosa, cervical lymph nodes (CLNs) and peripheral blood of the Tg mice, and displayed a polarization toward a nonprotective Th2 response. Proliferation of CLN CD4+ T cells from infected Tg mice in response to C. albicans Ag in vitro was abrogated and the cells failed to acquire an effector phenotype. Coculture of C. albicans-pulsed DCs with CD4+ T cells in vitro showed that Tg expression in either or both of these cell populations sharply reduced the proliferation of CD4+ T cells and their production of IL-2. Finally, transfer of naive non-Tg CD4+ T cells into these Tg mice restored proliferation to C. albicans Ag and sharply reduced oral burdens of C. albicans. Overall, these results indicate that defective CD4+ T cells primarily determine the susceptibility to chronic carriage of C. albicans in these Tg mice.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Studies report oropharyngeal candidiasis (OPC)3 is the most frequent opportunistic fungal infection among HIV-infected patients (1). Although the incidence of OPC in HIV infection has been significantly reduced since the introduction of highly active antiretroviral therapy (HAART) (2), it remains a common opportunistic infection in HIV-infected patients worldwide.

Although an intact adaptive cell-mediated immune response to Candida albicans is protective against OPC (3, 4), the critical impairments of innate and adaptive immunity that are responsible for the onset and maintenance of mucosal candidiasis in HIV infection remain undefined (reviewed in Ref. 5). Colonization of oral mucosal surfaces and symptomatic disease are closely correlated with progression of the cellular immunodeficiency of HIV infection (6). Although deficiencies in Candida-specific systemic cell-mediated immunity do not solely account for susceptibility to OPC in HIV-infected patients (7), the devastating impact of HIV infection on mucosal Langerhans’ cells (8, 9, 10) and CD4+ T cell (10) populations is most likely central to the pathogenesis of mucosal candidiasis in HIV-infected patients (reviewed in Ref. 5).

Controlled studies on the immunopathogenesis of mucosal candidiasis in HIV infection have been hampered by the lack of a relevant animal model (11) (reviewed in Ref. 5). The availability of CD4C/HIV transgenic (Tg) mice expressing gene products of HIV-1 in immune cells and developing an AIDS-like disease has provided an opportunity to devise a novel model of mucosal candidiasis (12). These CD4C/HIV Tg mice are immunodeficient and exhibit atrophy of lymphoid organs, a preferential depletion of CD4+ T cells, with altered CD4+ T cell proliferation in vitro, loss of CD4+ T cell help, CD4+ T cell and B cell activation and impaired dendritic cell (DC) function (13, 14, 15, 16, 17). In addition, disease of the lung (lymphocytic interstitial pneumonitis), heart (myocytolysis, myocarditis), and kidney (tubulointerstitial nephritis, segmental glomerulosclerosis, microcystic dilatation) develop in these Tg mice (13, 18). We have previously reported that mucosal Candida infection in these Tg mice closely mimics the clinical and pathological features of candidal infection in human HIV infection (12) (reviewed in Ref. 5). With the recognition that a cause-and-effect analysis of the immunopathogenesis of mucosal candidiasis in HIV infection can now be achieved under controlled conditions in these Tg mice, the present study was undertaken to determine the role of defective DCs and CD4+ T cells in impaired induction of protective immunity and in the phenotype of chronic oral carriage of C. albicans. In this study, we show that depletion and functional impairment of these cell populations present in Tg mice abrogates Candida-specific adaptive immunity in vivo and in vitro, and that protective immunity to C. albicans can be reconstituted by transferring intact CD4+ T cells into Tg mice. Therefore, altered CD4+ T cells are central to the immunopathogenesis of mucosal candidiasis in these Tg mice expressing HIV-1.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Generation of Tg mice expressing HIV-1 gene products

The CD4C/HIVMutA Tg mice, which express Rev, Env, and Nef of HIV-1, have been described elsewhere (13). The CD4C/HIVMutA construct harbors mouse CD4 enhancer and human CD4 promoter elements to drive the expression of HIV-1 genes in CD4+CD8+ and CD4+ thymocytes, in peripheral CD4+ T cells, and in macrophages and DCs. Founder mouse F21388 was bred on the C3H background. Specific pathogen-free male and female Tg mice and non-Tg littermates were housed in sterilized individual cages equipped with filter hoods and were supplied with sterile water and mouse chow. In selected experiments, male and female 5- to 6-wk-old C3H mice (17–18 g; Charles River Laboratories) were used as immunocompetent controls. All animal experiments were approved by the Animal Care Committee of the University of Montreal.

Animal model of mucosal candidiasis

Oral inoculation with C. albicans strain LAM-1 assessments for signs of morbidity and quantification of C. albicans in the oral cavities of individual mice were conducted as previously described (12). In brief, mice were anesthetized with 0.1 ml of a solution containing 1 ml of Hypnorm (0.315 mg/ml fentanyl citrate and 10 mg/ml fluanisone; Janssen Pharmaceutica), 1 ml of Versed (5 mg/ml midazolam; Hoffmann-LaRoche), and 2 ml of water, i.p. They were then inoculated by topical application of 108 pelleted late-log phase blastoconidia recovered on sterile calcium alginate Calgiswabs (Fisher Scientific). A longitudinal quantification of C. albicans in the oral cavities of individual mice was done from day 1 until euthanizing of the animals. Calgiswabs used for sampling were dissolved in 2-ml volumes of Ringer’s citrate buffer, and serial dilutions were prepared in PBS and plated on Sabouraud dextrose agar supplemented with chloramphenicol (0.05 g/L). Plates were incubated for 24 h at 37°C.

Candida Ag

C. albicans strain LAM-1 was grown in Sabouraud dextrose broth (Difco) for 18 h at 30°C with rotary agitation. To prepare a crude cytoplasmic extract (3), yeast cells were washed twice in sterile 0.01 M PBS (pH 7.4), resuspended in PBS at 1.2 x 1010 cells/ml, and disrupted with 20 successive 20-s runs and intermittent cooling in a Vibra Cell instrument (Sonics & Materials) set at an amplitude of 20%. The cell lysate was centrifuged for 10 min at 2000 x g, and the supernatant was dialyzed against PBS. The extract (8.5 mg of protein/ml) was stored in aliquots at –20°C.

Single-cell suspensions of spleen, cervical lymph node (CLN), and oral mucosal tissue

Groups of 6–10 CD4C/HIVMutA and non-Tg littermates (45- to 55-day-old) were orally infected or not with 108 CFU of C. albicans LAM-1 blastoconidia (12) and assessed at 7, 45, or 70 days postinfection. Independent experiments were conducted by pooling cells from all mice within each group. Heparinized blood was collected by cardiac puncture under anesthesia with Hypnorm (Janssen Pharmaceutica) and Versed (Sabex) (12), and the mice were exsanguinated with PBS (12). Spleens and CLNs were removed, mechanically disrupted by pressing through a nylon mesh (pore size, 70 µm), and deposited in 25-mm diameter dishes containing 2 ml of HBSS (Invitrogen Life Technologies). Cell suspensions were washed twice in HBSS and resuspended in complete tissue culture medium consisting of RPMI 1640 (Invitrogen Life Technologies) supplemented with 10% of heat-inactivated FBS (Invitrogen Life Technologies), 20 mM HEPES buffer, 2 mM L-glutamine, 5 x 10–5 M 2-ME, 100 U/ml penicillin and streptomycin, 0.25 µg/ml amphotericin B, and 50 µg/ml gentamicin. Cells were filtered through a sterile nylon mesh (pore size, 70 µm) to obtain a homogeneous suspension.

The cheeks and the hard and soft palate were dissected free of the underlying muscle layer and washed for 5 min in cleaning buffer solution (20 mM Tris, 20 mM NaCl, 40 mM EDTA, and 1 mM DTT). Tissues were washed in HBSS, cut longitudinally, and minced into 1-mm2 fragments in complete medium. Minced tissues were digested by incubating with 0.25% collagenase type IV (Sigma-Aldrich) in complete medium at 37°C for 30 min with gentle agitation, replacing the medium, and incubating for a further 30 min. Tissue debris were excluded by twice filtering cell suspensions through a 70-µm nylon mesh.

Contamination of these cell suspensions by peripheral blood cells was <1% according to the RBC to nucleated cell ratio. Cell suspensions were resuspended in complete medium and adjusted to 1 x 106 cells/ml, and cell viability was >90% by trypan blue exclusion.

Flow cytometry

Cell surface marker analysis of PBLs was conducted as previously described (12) on a FACSCalibur flow cytometer (BD Biosciences) equipped with CellQuest software. CLN and oral mucosal cells were surface stained with different combinations of anti-mouse anti-CD45, anti-CD11b, anti-I-AK (MHC class II alloantigen), anti-CD11c, anti-CD3, anti-CD4, and anti-CD8 fluorescent-labeled mAbs, and their respective isotype controls (BD Biosciences). Cells were twice washed in cold PBS and fixed with 2% paraformaldehyde. For spleen cell suspensions, RBCs were removed with lysing buffer (BD Biosciences) before washing and fixation.

Spleen cells were used as a control for comparison of flow cytometry profiles with the oral mucosal cell population. Data were acquired on 10,000 events by gating on CD45+ cells, which comprised 6–8% of mucosal cell suspensions, and expressed according to the various combinations of Abs (Fig. 1). Collagenase treatment of splenocytes decreased the percentage of CD3+CD8+ T cells by an average of 28% but did not alter the percentage of CD3+CD4– cells, which remained equivalent to that of CD3+CD8+ cells untreated with collagenase. Accordingly, CD8+ T cells were estimated as CD3+CD4– cells in the oral mucosa. Treatment with collagenase did not change the percentage of CD3+CD4+ T cells.


Figure 1
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  		FIGURE 1. Flow cytometry analysis of oral mucosal cell populations in CD4C/HIVMutA Tg mice. Acquisition was gated on CD45+ cells from spleen and oral mucosa, and multicolor analysis was conducted to identify and quantitate specific cell populations. Immature and mature DCs were identified as CD11bhigh, I-Ak+, CD11c+ and CD11blow, I-Ak+, CD11c+, respectively, whereas CD4+ T cells were CD3+ and CD4+.

 
Determination of intracellular cytokines

Positive selection of CD4+ T cells from CLN cell suspensions was done using biotin-conjugated Ab specific for CD4 (BD Biosciences) and streptavidin Captivate ferrofluid particles (Molecular Probes), yielding >95% CD4+ T cells. No difference in the generated data was observed by using the Dynal Mouse CD4 Negative Isolation kit (Dynal Biotech). A total of 106 selected CD4+ T cells/ml was cultured for 72 h on anti-CD3-coated (5 µg/ml; BD Biosciences) and anti-CD28-coated (5 µg/ml; BD Biosciences) plates in supplemented RPMI 1640 containing 5 µg/ml anti-CD3 Ab. The cells were restimulated for 4 h with ionomycin (500 ng/ml) and PMA (5 ng/ml) in presence of monensin (GolgiStop; BD Biosciences) or brefeldin A (GolgiPlug; BD Biosciences), and surface stained with anti-CD69 Ab (BD Biosciences). After washing, intracellular staining was performed with the Cytofix/Cytoperm kit (BD Biosciences) and anti-mouse IFN-{gamma}-PE, anti-IL-2 FITC, anti-IL-4 allophycocyanin, anti-IL-10 FITC, anti-TNF-{alpha} PE, and their respective isotype control Abs (BD Biosciences). Flow cytometry analysis was performed by gating on CD69+ cells, to determine the percentage of CD4+ T cells expressing each of the cytokines.

Bone marrow-derived DCs (BMDCs) were generated using a modification of methods previously described (16, 19). BM cells were filtered through a 70-µm nylon mesh, washed with HBSS, and cultured with GM-CSF (1000 U/ml; Cedarlane Laboratories) and IL-4 (500 U/ml; Cedarlane Laboratories) in supplemented RPMI 1640. At days 2 and 3, nonadherent cells were removed and fresh medium supplemented with GM-CSF and IL-4 was added. Nonadherent cells obtained at day 7 were determined to be DCs by morphology and FACS analysis (>90% CD11b+CD11c+I-Ak+). A total of 106 BMDCs was stimulated with LPS (100 ng/ml or 1 µg/ml; Sigma-Aldrich) or pulsed with live C. albicans blastoconidia at different DC to Candida ratios for 2 h before addition of brefeldin A and 2.5 µg/ml amphotericin B to prevent Candida overgrowth, and incubated for a further 16 h. Amphotericin B was reported to not modify cytokine production by DCs (20). Intracellular staining was performed with anti-IL-12 PE (BD Biosciences), and the percentage of CD11c+ DCs expressing IL-12 was determined by flow cytometry.

Quantitative RT-PCR analysis of cytokines in oral mucosal tissue

Cheeks were collected into RNAlater (Qiagen) and stored at –20°C. Total RNA was extracted using the RNeasy Mini kit with tissue disruption using an Omni TH-115 homogenizer (Omni International), and DNase digestion as recommended by the manufacturer. The extracted RNA was quantitated using the RiboGreen RNA Quantitation Reagent and kit (Molecular Probes) and its integrity was confirmed by agarose gel electrophoresis. RNA (600 ng) was reverse-transcribed using the QuantiTect Reverse Transcription kit (Qiagen). cDNA was amplified in duplicate by real-time PCR in a SmartCycler instrument (Cepheid), using the QuantiTect SYBR Green PCR kit (Qiagen) and a user-developed protocol found at the manufacturer’s website (<www1.qiagen.com/literature/protocols/pdf/PCR04.pdf>). Primers for IL-4 were identified through a search in PrimerBank (ID no. 10946584a1) (21). Primers for IFN-{gamma} (forward: TCA AGT GGC ATA GAT GTG GAA GAA, reverse: TGG CTC TGC AGG ATT TTC ATG) and for beta-actin as a housekeeping gene (forward: AGA GGG AAA TCG TGC GTG AC, reverse: CAA TAG TGA TGA CCT GGC CGT) were identified through a search at the BLAST program web site (<www.ncbi.nlm.nih.gov>). Cytokine mRNA expression was normalized against the expression of beta-actin, and relative quantitation was performed using the comparative threshold (Ct) cycle number method available at the University of Freiburg web site (<www.ukl.uni-freiburg.de/core-facility/lc480/user_bulletin_2.pdf>). Comparative threshold of the target genes, normalized {Delta}Ct (Ctcytokine – Ctactin), {Delta}{Delta}Ct (average {Delta}Ct – average {Delta}Ctnon-Tg), and relative level (2–{Delta}{Delta}Ct) were calculated in Excel. Control experiments demonstrated that expression of beta-actin in cheek mucosa was not altered by HIV-1 transgene expression or candidal infection (data not shown).

Proliferative response of CLN CD4+ T cells to Candida Ag and Con A

CLN cells were stained with 1 µM CFSE dye (Molecular Probes) in the dark at 22°C for 8 min. Cells were washed three times with PBS containing 5% FBS and cultured in triplicate for 96 h in flat-bottom 96-well plates at 2 x 105 cells/well, containing Con A (5 µg/ml; Sigma-Aldrich), C. albicans Ag (66 µg/ml) or cells alone. These concentrations produced maximal proliferation of CLN cells from C3H mice, 7 days after oral infection with C. albicans. Then, the cells were surface stained with anti-CD4 PerCP and anti-CD62L PE Abs (BD Biosciences) and analyzed by flow cytometry. Cell proliferation was estimated as the percentage of CD4+ T cells with decreased CFSE fluorescence intensity (22), and decreased CD62L expression determined differentiation into CD4+CD62L– effector cells.

In vitro activation of CLN CD4+ T cells by Candida-pulsed DCs

Purified CLN CD4+ T cells (>90% CD3+CD4+ T cells) were isolated using the Dynal Mouse CD4 Negative Isolation kit (Dynal Biotech). DCs (2 x 104 cells), pulsed or unpulsed for 18 h with live C. albicans blastoconidia at a 1:5 ratio as described earlier, were cultured with 2 x 105 CFSE-labeled CD4+ T cells for 5 days in 100 µl of supplemented RPMI 1640 medium. Cell proliferation was estimated as the percentage of CD4+ T cells with decreased CFSE fluorescence intensity (22). IL-2 production in coculture supernatants was quantitated using the Mouse Cytokine Bead Array kit (BD Biosciences).

CLN activated/memory-like T cells

CLN cells were surface stained with anti-CD4 PerCP, anti-CD8 FITC, anti-CD44 biotin and anti-CD62L PE Abs (BD Biosciences), washed twice with PBS, and stained with streptavidin-allophycocyanin (BD Biosciences). After washing, flow cytometry analysis was performed by gating on CD4+ or CD8+ cells to quantitate the proportion of naive and memory-like CD4+ and CD8+ T cells (23).

C. albicans phagocytosis assay

Immediately before use, C. albicans yeast cells were heat inactivated at 95°C for 30 min and labeled with FITC (10 µg/ml; Sigma-Aldrich) in 0.5 M carbonate buffer (pH 9.5) for 60 min at 4°C. For the phagocytosis assay, 2.5 x 105 BMDCs were incubated with C. albicans cells at different DC to Candida ratios in supplemented RPMI 1640 at 37°C. Phagocytosis was stopped by cooling the samples to 4°C. A control assay was conducted at 4°C (1:5 ratio) in presence of cytochalasin B (5 µg/ml; Sigma-Aldrich) (24). Ethidium bromide (10 µg/ml) was added and the percentage of DCs with phagocytosed C. albicans was determined by flow cytometry (25). Phagocytosis of C. albicans by DCs was verified by confocal microscopy.

Adoptive transfer of DCs and CD4+ T cells

BMDCs and purified CLN CD4+ T cells were obtained from uninfected non-Tg mice as described. A total of 5 x 105 DCs and/or 2.5 x 106 CD4+ T cells in 100 µl of PBS, or PBS alone, was injected i.v. into C. albicans-infected Tg mice at days 21 and 27 after oral infection with C. albicans, respectively, in the early phase of chronic oral carriage. Oral burdens of C. albicans were serially monitored after transfer, and CLN CD4+ T cell proliferation was measured at the conclusion of the observation period.

Statistical analysis

Differences in oral burdens of C. albicans among groups of mice were analyzed using PROC MIXED software (SAS Institute). Repeated-measurement ANOVA with Tukey-Kramer contrast analysis when required was conducted with two factors, one between (group) and one within (time). Significant interactions (p < 0.05) were further analyzed, and significant differences (p < 0.05) between group means at fixed times were determined by use of the two-sample, two-tailed Student’s t test for independent samples. Immunophenotypes of cell populations and cytokine expression by Tg mice determined on the day of infection were analyzed using Student’s t test. Statistical analyses for all other data were performed with SPSS version 11.5 software, using an ANOVA followed by Tukey contrast analysis when necessary. Differences were considered to be significant at values of p < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Immunophenotypes of oral mucosal and CLN cell populations

To longitudinally assess the impact of the HIV-1 transgene on DCs, and CD4+ and CD8+ T cell populations, multiparametric flow cytometry analysis was conducted on CD4C/HIVMutA Tg mice and non-Tg littermates infected or not with C. albicans. In comparison to non-Tg littermates, CD4C/HIVMutA Tg mice had a strikingly depleted CD11blow, I-AK+, CD11c+ mature DC population in the oral mucosa throughout the course of AIDS-like disease (p < 0.05), which did not progress over time (p > 0.05) (Figs. 2A and 3). Oral infection with C. albicans did not alter the percentage of these mature DCs in the Tg mice (p > 0.05), but resulted in a sharp decrease (p < 0.001) of this cell population 7 days after oral infection in the non-Tg animals (Fig. 2A). A similar transient decrease (p = 0.006) in mature DCs coinciding with recovery from primary oral infection with C. albicans was observed in the oral mucosae of C3H mice (our unpublished data). In contrast to mature DCs, no depletion of the CD11bhigh, I-AK+, CD11c+ immature DC population appeared during the course of AIDS-like disease in these Tg mice (p > 0.05), and oral infection with C. albicans did not alter the percentage of these cells in either Tg or non-Tg animals (p > 0.05). A depletion in oral mucosal CD4+ T cells in uninfected Tg compared with non-Tg mice appeared early (p = 0.01) and remained constant throughout AIDS-like disease (Figs. 2A and 3). Both Tg and non-Tg mice were able to respond to C. albicans infection with an increase (p < 0.05) in oral mucosal CD4+ T cells; however, CD4+ T cells remained lower (p < 0.001) in infected Tg compared with infected non-Tg mice (Figs. 2A and 3). Finally, oral mucosal CD8+ T cells, estimated as CD3+CD4– cells, were augmented (p < 0.05) in infected Tg mice compared with uninfected non-Tg animals both early and late in the course of AIDS-like disease, demonstrating that the Tg mice recruit this cell population to the oral mucosa in response to candidal infection (Fig. 2A).


Figure 2
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  		FIGURE 2. Longitudinal assessment of immunophenotypes of oral mucosal (A), CLN (B), and peripheral blood cell populations (C) in CD4C/HIVMutA Tg mice infected or uninfected with Candida albicans. Data are presented as the percentage of CD45+ cells or as absolute numbers of cells, and are the mean ± SD of three to nine independent experiments. Significantly different (*, p < 0.05) from non-Tg mice and (**, p < 0.05) from uninfected mice.

 

Figure 3
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  		FIGURE 3. Multicolor analysis of oral mucosal DCs and CD4+ T cells in CD4C/HIVMutA Tg mice infected or uninfected with Candida albicans. Representative analyses demonstrate the striking depletion of mature CD11blow,I-Ak+,CD11c+ DCs 7 days after infection with C. albicans (top) and of CD4+ T cells in these Tg mice 45 days after infection with C. albicans (bottom).

 
In contrast with the oral mucosa, the proportion of CLN I-AK+,CD11c+ DCs was augmented (p < 0.01) in the Tg compared with the non-Tg mice (Fig. 2B), but was not significantly altered by oral infection with C. albicans (p > 0.05). However, because of the hypocellularity of CLNs (Fig. 2B) and of the secondary lymphoid organs in these Tg mice (13), the absolute numbers of CLN DCs were actually markedly decreased in the Tg compared with the non-Tg mice (p < 0.05) (Fig. 2B). In contrast with the oral mucosa and CLNs, however, the percentage of peripheral blood DCs was unaffected by expression of the transgene or infection with C. albicans (p > 0.05) (our unpublished data). As in the oral mucosa, CLN CD4+ T cells were sharply depleted in the Tg compared with the non-Tg mice (p < 0.001) (Fig. 2B), as documented previously (13, 15). In contrast with the oral mucosa, however, oral infection with C. albicans did not change the proportion of CD4+ T cells in the CLNs of the Tg mice (p > 0.05), but resulted in a sustained increase of this cell population in the CLNs of the non-Tg mice (p < 0.05) (Fig. 2B). The mean fluorescence of CLN CD4+ T cells from the Tg mice was significantly reduced (p < 0.001) to only 60% of values measured in cells from non-Tg mice, throughout AIDS-like disease, as previously observed (13). This CD4 down-regulation was observed irrespective of infection with C. albicans (p > 0.05) (our unpublished data). The percentage of CD8+ T cells was augmented in the CLNs of the infected Tg compared with the non-Tg mice before and until 7 days after infection (p < 0.02), but was comparable in all four groups of mice during the later period of observation (p > 0.05) (Fig. 2B). Expressed as absolute numbers, however, CLN CD8+ cells were depleted in the Tg compared with the non-Tg mice (p < 0.05), but increased (p < 0.05) in both groups of mice 7 days after oral infection with C. albicans (Fig. 2B). The mean fluorescence of these CLN CD8+ T cells was also decreased (p < 0.001) to 67% of values found in cells from the non-Tg mice, both early and late in the course of AIDS-like disease and independently of infection with C. albicans (p > 0.05) (our unpublished data). Peripheral blood CD4+ and CD8+ T cells were also severely depleted in the Tg compared with the non-Tg mice throughout AIDS-like disease (Fig. 2C).

Oral mucosal DCs from Tg mice displayed a lower (p < 0.05) expression of MHC class II (I-AK) molecules compared with non-Tg mice, thus further compounding the immune defect in these Tg mice (Fig. 2A). As reported before (16), surface expression of MHC class II was also sharply decreased on CLN I-AK+, CD11c+ DCs from the Tg mice (p < 0.05) (Fig. 2B). However, MHC class II expression on oral mucosal and CLN DCs from Tg or non-Tg mice was unaltered by oral infection with C. albicans (p > 0.05) (Fig. 2, A and B).

Expression of cytokines by CLN CD4+ T cells and BMDCs, and cytokine gene expression in oral mucosal tissue

To determine the effect of the HIV transgene on Th1 and Th2 responses, the expression of cytokines by CLN CD4+ T cells was compared in Tg and non-Tg mice infected or uninfected with C. albicans. The percentages of CD4+ T cells producing the Th1 cytokines IL-2 and TNF-{alpha} and the Th2 cytokine IL-10 were all augmented (p < 0.05) in the Tg compared with the non-Tg mice throughout AIDS-like disease (Fig. 4A). In addition, expression of IL-10 in the Tg but not the non-Tg mice was further enhanced by oral infection with C. albicans (p < 0.05), in contrast to the expression of IL-2 and TNF-{alpha}, which was unaltered by infection in both Tg and non-Tg mice (p > 0.05). Percentages of CD4+ T cells producing the Th1 cytokine IFN-{gamma} were also augmented early (p = 0.003) and until day 45 in the Tg compared with the non-Tg mice, but fell precipitously by day 70 to a level significantly lower (p < 0.001) than at the earlier time points. In contrast, no such change in expression over time was observed in the non-Tg mice (p > 0.05), and oral infection with C. albicans did not alter expression of IFN-{gamma} in either the Tg or the non-Tg mice (p > 0.05). Finally, the percentages of cells producing the Th2 cytokine IL-4 were comparable in the four groups of mice until day 45 (p > 0.05), but rose markedly in the Tg mice infected with C. albicans compared with infected or uninfected non-Tg mice (p = 0.025) on day 70. Expression of IL-4 in these Tg mice infected with C. albicans was significantly greater on day 70 compared with earlier time points (p < 0.01). Taken together, these results indicate a partial polarization of CLN CD4+ T cells toward a Th2 response during progression of AIDS-like disease in these Tg mice orally infected with C. albicans. Because IL-12 promotes CD4+ T cell differentiation into CD4+ Th1 cells (26), we next measured the production of IL-12 by BMDCs. In comparison to non-Tg mice, DCs generated from the Tg mice with GM-CSF and IL-4 and pulsed with live C. albicans blastoconidia had a reduced capacity to produce IL-12 (Fig. 4B), indicating that defective priming of Th1 cells in the Tg mice may at least partially result from defective production of IL-12 by DCs.


Figure 4
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  		FIGURE 4. A, Expression of cytokines by CLN CD4+ T cells in CD4C/HIVMutA Tg and non-Tg mice infected or uninfected with Candida albicans. Data are mean ± SD of three to five independent experiments. Significantly different (*, p < 0.05) from non-Tg mice and (**, p < 0.05) from uninfected mice. B, Percentages of BMDCs from CD4C/HIVMutA Tg and non-Tg mice expressing IL-12. DCs were stimulated with LPS or live C. albicans blastoconidia at the indicated ratios. Data are mean ± SD of three to eight independent experiments. Significantly different (*, p < 0.01) from non-Tg mice. C, Expression levels (2–{Delta}{Delta}Ct) of IFN-{gamma} and IL-4 in oral mucosal tissues of CD4C/HIVMutA Tg and non-Tg mice, normalized to the endogenous control gene beta-actin and relative to control non-Tg mice uninfected with Candida albicans. Data are mean ± range of five to six mice at each time point.

 
In comparison with uninfected non-Tg mice, the relative expression of IFN-{gamma} mRNA in whole oral mucosal tissue was augmented more than 2-fold in the Tg mice on day 70, irrespective of infection with C. albicans (Fig. 4C). Expression of IFN-{gamma} on day 70 was also increased in infected non-Tg mice, demonstrating that transgene expression and candidal infection both independently augmented expression of this cytokine in the oral mucosa at this late stage in AIDS-like disease. Early after primary infection with C. albicans (day 7), however, both transgene expression and candidal infection were required to produce a >2-fold increase in expression of IFN-{gamma} relative to uninfected non-Tg mice (Fig. 4C). In comparison with uninfected non-Tg mice, the relative expression of IL-4 mRNA was increased >2-fold on day 7 in both infected and uninfected Tg mice, as well as infected non-Tg mice (Fig. 4C). Therefore, transgene expression and Candida infection independently enhanced the expression of IL-4 early in AIDS-like disease. However, by day 70, the relative expression of IL-4 in the oral mucosa of the Tg mice was comparable to that in the uninfected non-Tg mice (Fig. 4C).

Proliferative response of CLN CD4+ T cells

CLN CD4+ T cells harvested from non-Tg mice 7 days after infection proliferated in vitro in response to C. albicans Ag and acquired an effector phenotype (CD4+CD62L–), whereas the cells from the infected Tg mice failed to proliferate and did not acquire an effector phenotype (CD4+CD62L+) (Fig. 5A). Because the percentage of I-AK+, CD11c+ CLN DCs was modestly increased in the Tg (4%) compared with the non-Tg mice (3%) (Fig. 2B), the impaired in vitro proliferative response to C. albicans Ag in the Tg mice did not result from a lower number of DCs in the assay. Although infected and uninfected Tg mice maintained proliferative responses to Con A, these were lower (p < 0.05) in the Tg compared with the infected and uninfected non-Tg mice, respectively (Fig. 5A). In addition, oral infection with C. albicans increased the response of the non-Tg (p = 0.004) but not that of the Tg animals (p > 0.05) to Con A. Accordingly, the proliferative response to Con A was diminished in the Tg compared with the non-Tg mice independently of infection with C. albicans.


Figure 5
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  		FIGURE 5. A, Reduced proliferative response of CLN CD4+ T cells to Con A and Candida albicans Ag (Ag) in CD4C/HIVMutA Tg compared with non-Tg mice 7 days after infection with C. albicans (top). Data are mean ± SD of three and four to six independent experiments, respectively. CFSE-labeled CLN CD4+ T cells from non-Tg mice harvested 7 days after infection proliferated in vitro in response to C. albicans Ag and acquired an effector phenotype (CD4+CD62L–), whereas cells from the infected Tg mice failed to proliferate and did not acquire an effector phenotype (CD4+CD62L+) (bottom). Significantly different (*, p < 0.05) from non-Tg mice and (p < 0.05) from uninfected mice. B, Proliferative responses of isolated CLN CD4+ T cells from CD4C/HIVMutA Tg or non-Tg mice infected or uninfected with Candida albicans, following coculture with BMDCs from Tg or non-Tg mice pulsed or unpulsed with live C. albicans yeast cells. In comparison to non-Tg mice, DCs and CD4+ T cells isolated from the Tg mice independently produced an impaired proliferative response to C. albicans (top). IL-2 production in supernatants of CD4+ T cell/DC cocultures (bottom). HIV-1 transgene expression in either or both of these cell populations sharply reduced production of IL-2. Infection with C. albicans augmented but did not fully restore production of the cytokine to levels found in the non-Tg mice. Data are mean ± SD of 3–12 independent experiments. Significantly different (*, p < 0.01) from the respective non-Tg CD4 control and (**, p < 0.001) from the respective non-Tg DC control. Significantly different (***, p < 0.001) from the respective CD4 control from uninfected mice. C, Phagocytosis of C. albicans yeast cells by BMDCs from CD4/HIVMutA Tg and non-Tg mice. Analyses were performed at the indicated DC to yeast cell ratio. Data are mean ± SD of three independent experiments.

 
In vitro activation of CLN CD4+ T cells by Candida-pulsed DCs

When cocultured with unpulsed DCs from Tg or non-Tg mice, CLN CD4+ T cells isolated from infected or uninfected Tg mice had a consistently reduced ability to proliferate compared with CD4+ T cells from non-Tg mice (p < 0.01) (Fig. 5B). The reduced proliferation of CD4+ T cells from the Tg mice was not altered (p > 0.05) by infection with C. albicans and was not rescued by coculture with unpulsed DCs from non-Tg mice (p > 0.05). Furthermore, Candida-pulsed DCs from Tg mice were uniformly defective in their ability to activate CD4+ T cells from infected or uninfected Tg and non-Tg mice (p < 0.001) (Fig. 5B). This result is consistent with a previous report showing impaired capacity of Tg DCs to present both cytochrome c protein and peptide to pigeon cytochrome c-specific CD4+ T cells (16). Therefore, defective DCs and CD4+ T cells from the Tg mice independently contributed to the impaired proliferation of CD4+ T cells in vitro, which was further demonstrated by a sharply reduced production of IL-2 in coculture supernatants (Fig. 5B). Nevertheless, infection with C. albicans enhanced the proliferation of CD4+ T cells on coculture with Candida-pulsed DCs (p < 0.001), independently of the source of CD4+ T cells and DCs from Tg or non-Tg mice, and hastened peak IL-2 production from 120 to 24 h of coculture (Fig. 5B).

Phagocytosis of C. albicans by DCs

Phagocytosis of C. albicans yeast cells by BMDCs from Tg and non-Tg mice was compared to determine whether defective uptake of the fungus by DCs from the Tg mice contributes to their perturbed activation of CD4+ T cells. No significant differences (p > 0.05) in the percentage of DCs with ingested C. albicans were found between the Tg and non-Tg mice, measured sequentially over 120 min and at three different DC:yeast cell ratios (Fig. 5C). Perturbations of DCs that impact on the induction of adaptive immunity to C. albicans in the Tg mice are therefore likely located further downstream, after phagocytosis of the fungus.

Memory-like phenotype of CLN CD4+ and CD8+ T cells

It was previously reported that CD4+ T cells from CD4C/HIVMutG Tg mice exhibit an activated memory-like phenotype that appears to be independent of antigenic stimulation (15). Consistent with this, we found that in the absence of in vitro antigenic or mitogenic stimulation, the proportion of naive CLN CD4+ T cells (CD44–/low, CD62L+) was decreased (p < 0.05) and the proportion of activated CD4+ memory-like T cells (CD44high, CD62L–) increased (p < 0.01) in the Tg compared with the non-Tg mice infected or uninfected with C. albicans, both early and late during the course of AIDS-like disease (Fig. 6). However, the proportion of resting CD4+ T-memory-like cells (CD44high, CD62L+) was unaltered in these Tg mice (p > 0.05). Oral infection with C. albicans did not change the proportion of naive, resting memory-like or activated memory-like CD4+ T cells in the Tg or non-Tg mice (p > 0.05). When calculated as absolute numbers, however, CLN naive, resting and activated memory cells were all depleted in the Tg compared with the non-Tg mice 70 days after infection with C. albicans (Fig. 6). Furthermore, candidal infection significantly enhanced the numbers of naive but not memory-like CLN CD4+ T cells in both Tg and non-Tg mice both early and late in AIDS-like disease (Fig. 6). Therefore, despite their enhanced proportion, CLN CD4+ T cells with an activated/memory-like phenotype were depleted due to the hypocellularity of secondary lymphoid organs in these Tg mice.


Figure 6
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  		FIGURE 6. Naive and memory-like CD4+ and CD8+ T cells in the CLNs of CD4C/HIVMutA Tg mice infected or uninfected with Candida albicans, expressed as the percentage or as the absolute numbers of cells. Data are mean ± SD of three independent experiments at each time point. Significantly different (*, p < 0.05) from non-Tg mice and (**, p < 0.05) from uninfected mice. Significantly different (***, p < 0.05) from the three other groups of mice.

 
In contrast with CD4+ T cells, the proportions of CLN naive and memory-like CD8+ T cells were comparable in Tg and non-Tg mice infected or uninfected with C. albicans, both early and at the later stage of AIDS-like disease (p > 0.05) (Fig. 6). However, as for CD4+ T cells, absolute numbers of naive and memory-like CLN CD8+ T cells were sharply decreased in the Tg compared with the non-Tg mice, but were significantly enhanced by C. albicans infection only in the non-Tg mice (Fig. 6). Altogether, these findings indicate a broad depletion of both naive and memory-like CD4+ and CD8+ T cells in these Tg mice.

Transfer of DCs and CD4+ T cells

Naive CD4+ T cells and DCs isolated from non-Tg mice were transferred either alone or combined into Tg mice with persistent oral carriage of C. albicans. Transfer of CD4+ T cells alone produced a striking reduction of oral burdens of C. albicans to levels one-fifth to one-half of those in sham-transferred Tg mice (p < 0.02), sustained over 8 days following transfer (Fig. 7A). In contrast, oral burdens of C. albicans were not significantly altered (p > 0.05) over 4 wk following transfer of DCs alone into these Tg mice (Fig. 7B). Consistent with these findings, transfer of DCs did not significantly change (p > 0.05) oral burdens of C. albicans in these Tg mice before transfer of CD4+ T cells 6 days later (Fig. 7C), but the latter transfer was followed by a sharp and sustained reduction of oral fungal burdens in comparison with sham-transferred Tg mice (p < 0.001) (Fig. 7C), comparable with that observed after transfer of CD4+ T cells alone (Fig. 7A). Finally, 8.4 ± 0.3% of CLN CD4+ T cells proliferated in response to C. albicans Ag 13 days after transfer of CD4+ T cells into these Tg mice with persistent oral candidiasis, in comparison with 1.9 ± 1.6% in sham-transferred Tg mice (p < 0.05; four mice per group), demonstrating a substantial restoration of adaptive immunity to C. albicans in comparison with infected non-Tg mice (Fig. 5A).


Figure 7
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  		FIGURE 7. Adoptive transfer of naive CD4+ T cells and DCs isolated from non-Tg mice, to CD4C/HIVMutA Tg mice orally infected with C. albicans. Chronic oral carriage of C. albicans was sharply reduced following transfer of CD4+ T cells alone or with DCs. Data are mean ± SD of four to nine mice. Significantly different (*, p < 0.05) from controls.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The results of this study demonstrate that 1) quantitative depletion and functional impairments of both DCs and CD4+ T cells prevent the priming of a protective T cell-mediated adaptive immune response to C. albicans in CD4C/HIVMutA Tg mice; and 2) transfer of CD4+ T cells into these Tg mice reconstitutes protective immunity against chronic oral carriage of C. albicans. Perturbations of CD4+ T cells are therefore central to the immunopathogenesis of persistent oral candidiasis in these HIV-1 Tg mice.

Expression of the HIV-1 transgene resulted in a profound depletion and impaired maturation of DCs at the site of primary uptake of Candida Ags in the oral mucosa, consistent with the previously observed defective maturation of lymph node DCs from these Tg mice (16). Defective maturation of DCs may have resulted from a direct effect of HIV-1 gene products, most probably Nef, or from indirect effects on DCs from an impaired T cell environment (16). The nef gene has been reported to down-modulate surface expression of MHC class II Ags and to induce intracellular accumulation of MHC class II-peptide complexes (27). Consistent with these impairments of DCs in the Tg mice, defective terminal differentiation of oral Langerhans’ cells was demonstrated in human HIV infection by decreased expression of MHC class II Ags (9, 10), as well as the presence of blunt dendrites, limited development of organelles, and lack of Birbeck granules (10). Furthermore, numbers of both oral (8, 28) and esophageal (29) Langerhans’ cells are depleted in HIV infection.

Tg expression did not alter the ingestion of C. albicans yeast cells by DCs, a critical first step required to activate DCs for IL-12 production needed to prime Th1 cells and protective immunity (20). Nevertheless, the CD4C/HIVMutA transgene reduced the percentage of DCs expressing IL-12, consistent with the impaired maturation of BMDCs from CD4C/HIVMutA Tg mice (16). Expression of the HIV env gene product gp120 in APCs and impaired CD40L induction on CD4+ T cells activated by Ag have been shown to impair IL-12 and IFN-{gamma} production by APCs, thereby preventing a protective Th1 response (30, 31). Interestingly, the CD4C/HIVMutA Tg mice express Env, Rev, and Nef of HIV-1, and CD4+ T cells from these mice express low levels of CD40L (14). Because CD40-CD40L interactions are required for IL-12 production, persistence and APC capacity of DCs (32, 33) and T cell activation is a prerequisite for DC maturation (34), impaired numbers and activation of CD4+ T cells found in the Tg mice may have contributed to these defective DC functions. The polarization toward a nonprotective Th2 response of CD4+ T cells that we found in these Tg mice may also have resulted from impaired IL-12 responsiveness due to a loss of IFN-{gamma} production, which is required to override the IL-4-induced inhibition of IL-12R beta2 expression (35). IFN-{gamma} is required for IL-12 responsiveness in mice with C. albicans infection (36), but production of this cytokine by CLN CD4+ T cells fell sharply in the Tg mice late in the course of AIDS-like disease. Furthermore, the mature DC population that was depleted in these Tg mice is known to induce high concentrations of the Th1 cytokines IFN-{gamma} and IL-2 but little or no Th2 cytokines, whereas the preserved immature DC population described before (16) induces large amounts of the Th2 cytokines IL-4 and IL-10 (37). The enhanced production of IL-4 and IL-10 by CLN CD4+ T cells found in these Tg mice is closely associated with a nonprotective immune response to C. albicans (38).

The enhanced expression levels of IFN-{gamma} in oral mucosal tissues of infected or uninfected Tg mice late in AIDS-like disease are consistent with the augmented expression of this cytokine in the oral mucosa of HIV-infected patients with or without OPC (39). Because CD4+ T cells are severely depleted in the oral mucosa of these Tg mice, other oral cell populations most likely account for the enhanced production of this Th1 cytokine. However, the enhanced expression of IFN-{gamma} in the oral mucosa of the Tg mice was insufficient to prevent chronic oral carriage of C. albicans. The lack of a nonprotective Th cytokine profile found in the oral mucosa of these Tg mice concurs with observations in HIV-infected patients, which failed to show a consistent Th cytokine dichotomy (39).

Because both human (40) and murine (20) DCs play a critical role in the initiation of an adaptive cell-mediated immune response to C. albicans (reviewed in Ref. 5) by their capacity to recognize, phagocytose and degrade Candida, and subsequently present Candida Ags to T cells, these combined defects of DCs in the Tg mice likely perturb presentation of Candida Ags and reduce the differentiation of CD4+ T cells into a protective Th1 phenotype. DCs from the Tg mice appear to process Ags normally, but show low expression levels of MHC class II, CD40 and CD86, resulting in impaired delivery of costimulatory signals and of the capacity to present pigeon cytochrome c to AD10 TCR CD4+ T cells (16). MHC class II (41, 42, 43) and costimulatory molecules (43) are both directly involved in Candida-specific T cell activation by APCs. Accordingly, loss of MHC class II and costimulatory molecule expression on DCs from the Tg mice are likely to cause impaired presentation of C. albicans Ags and to account at least in part for the dramatically reduced proliferation of CD4+ T cells from infected Tg mice in response to C. albicans Ag. In human HIV infection, expression of MHC class II (44) and costimulatory molecules CD80 and CD86 (45), formation of MHC class II-Ag complexes (44), and Ag presentation capacities of APCs and their ability to stimulate CD4+ T cell proliferation are all impaired (46, 47).

HIV nef expression caused a profound and persistent loss of CD4+ T cells in the CLNs and peripheral blood of these Tg mice (13), strikingly similar to CD4+ T cell depletion in HIV disease (reviewed in Refs. 48, 49). We confirmed these results and show in this study that CD4+ T cells are also depleted in the oral mucosa. The expression of nef in CD4C/HIVMutA and CD4C/HIVMutG mice has been shown to down-regulate the CD4 cell surface molecule, enhance apoptosis of peripheral CD4+ T cells independently of the Fas pathway, and induce chronic activation of CD4+ T cells (15, 17). The proportion of CLN CD4+ T cells with an activated/memory-like phenotype was increased in the Tg compared with the non-Tg mice (15), indicating persistent immune activation that may have contributed to exhaustion of the naive T cell pool (48, 49). However, infection with C. albicans did not independently alter the proportion of activated/memory-like CD4+ T cells in these Tg mice, congruent with the observation that up-regulation of activation occurs independently of stimulation by Ags through the TCR (15).

CD4+ T cells from the Tg mice had an impaired proliferative response and production of IL-2 in comparison with cells from non-Tg mice, upon coculture with pulsed or unpulsed DCs from non-Tg mice. In addition, proliferation of CD4+ T cells from the Tg mice was also reduced in response to Con A, which stimulates proliferation of T cells directly and therefore does not require DCs (50). Taken together, these results indicate that the reduced proliferative response of CD4+ T cells from Tg mice to C. albicans involves defects not only of DCs but also of the CD4+ T cells themselves. The present findings concur with the limited capacity of CD4+ T cells from CD4C/HIVMutG Tg mice, which express only nef, to divide in response to stimulation with anti-CD3 and anti-CD28 or in allogeneic MLR (15, 16). Among its multiple points of interference with CD4+ cellular mechanisms (reviewed in Ref. 51), the Nef protein interacts with several signaling molecules and thereby inhibits activation pathways required for cell proliferation. Nef not only interferes with activation of p56lck and as a consequence signaling via the IL-2R (52), but also hinders cell cycle progression by down-regulating cyclins D1 and A (53). Reduced production of IL-2 by CD4+ T cells from the Tg mice may also have contributed to defective proliferation of these cells in vitro because T cells require IL-2 to complete the late portion of the G1 phase and enter the S phase of the cell cycle (54). Proliferative responses to mitogens (55), C. albicans Ags (56), and stimulation with anti-CD3 (57) are all impaired in patients infected with HIV, accompanied by impaired induction of the early activation marker CD69 (57) and diminished IL-2 production (58).

A sharply reduced proliferative response was found on coculture of Tg DCs and Tg CD4+ T cells, compared with non-Tg DCs and Tg CD4+ T cells. These in vitro findings suggest that defective Tg DCs potentiate the qualitative and quantitative CD4+ defects in these Tg mice beyond a critical threshold, resulting in anergy of in vivo-harvested CLN CD4+ T cells to C. albicans Ag and persistent candidiasis. However, transfer of naive non-Tg CD4+ T cells alone into Tg mice restored proliferation to C. albicans Ag and reduced oral burdens of C. albicans, suggesting that Tg APCs maintain a partial functional capacity to present Ags, as supported by the partially preserved proliferative response of non-Tg CD4+ T cells cocultured with Tg DCs. Nevertheless, transfer of naive non-Tg DCs alone into these CD4C/HIVMutA Tg mice did not reduce oral carriage of C. albicans. It is unlikely that this lack of protection resulted from an insufficient number of transferred DCs, since an identical number of DCs protected mice against an i.v. challenge of C. albicans (20). Alternately, transferred DCs may not have successfully migrated to the oral mucosa to endocytose C. albicans, or failed to navigate toward CLNs for Ag presentation to CD4+ T cells. Taken together, however, these results indicate that the onset and maintenance of chronic oral candidiasis in these Tg mice is primarily determined by defects of CD4+ T cells. The results further emphasize the critical role of adaptive T cell-mediated immunity in protection against OPC (reviewed in Ref. 5), as previously shown by decreased C. albicans oral colonization after reconstitution of immunodeficient BALB/C and CBA/CaH nu/nu mice with naive CD4+ T cells (4).

Despite their progressive depletion in the Tg mice, CD8+ T cells maintained the ability to respond to C. albicans infection by a quantitative increase in both oral mucosa and CLNs. CD8+ T cells accumulate in the basal epithelial layer of the oral mucosa in HIV-infected patients with OPC, demonstrating that these cells can be actively recruited to the mucosa in response to candidiasis (10, 59). However, the precise role of CD8+ T cells in mucosal containment of C. albicans in HIV infection, either by direct growth inhibition of Candida, or more likely, by an indirect mechanism, remains to be determined (59). HIV-1 nef down-regulates MHC class I expression on human DCs (60, 61) and on lymph node DCs of CD4C/HIVMutA Tg mice (16), impairs presentation of Ags by DCs to CD8+ T cells (60), and affects the functional competence of CD8+ T cells (61, 62) Furthermore, absolute numbers of CLN memory-like CD8+ T cells were sharply decreased in the Tg mice. Accordingly, further work will be needed to fully assess the role of CD8+ T cells in the immunopathogenesis of oral candidiasis in these Tg mice.


   Acknowledgments
 
We thank Marie-Andrée Laniel for support in maintaining the transgenic mouse colony; Jacques Thibodeau and Johanne Poudrier for helpful discussions and advice; Miguel Chagnon, Michel Lamoureux, and Yves Lepage for statistical analysis; and Sylvie Julien and Claire St.-Onge for manuscript preparation.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


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

1 This study was supported by Grant HOP-41544 from the Canadian Institutes of Health Research, HIV/AIDS Research Program. D.L. and M.M. are recipients of studentship awards from the University of Montreal and the Medical Mycology Research Fund of the University of Montreal, respectively. A.-C.L.-M. is the recipient of a Summer Student award from the Fonds de la Recherche en Santé du Québec. Back

2 Address correspondence and reprint requests to Dr. Louis de Repentigny, Department of Microbiology and Immunology, Sainte-Justine Hospital, 3175 Côte Ste-Catherine, Montreal, Quebec H3T 1C5, Canada. E-mail address: louis.de.repentigny{at}umontreal.ca Back

3 Abbreviations used in this paper: OPC, oropharyngeal candidiasis; Tg, transgenic; DC, dendritic cell; CLN, cervical lymph node; BMDC, bone marrow-derived DC. Back

Received for publication October 19, 2005. Accepted for publication April 17, 2006.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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