INFECTION AND IMMUNITY, Feb. 2002, p. 724–731
Copyright © 2002, American Society for Microbiology. All Rights Reserved.
Vol. 70, No. 2
Primary Role for CD4?T Lymphocytes in Recovery from
C. S. Farah,1* S. Elahi,2K. Drysdale,1G. Pang,2T. Gotjamanos,3G. J. Seymour,1
R. L. Clancy,2and R. B. Ashman1
Oral Biology and Pathology, School of Dentistry, University of Queensland, Brisbane, Queensland 4072,1Discipline of Immunology
and Microbiology, University of Newcastle, Newcastle, New South Wales 2300,2Department of Pathology, QEII Medical Centre,
University of Western Australia, Nedlands, Western Australia 6907,3Australia
Received 23 July 2001/Returned for modification 9 August 2001/Accepted 8 November 2001
Oropharyngeal candidiasis is associated with defects in cell-mediated immunity and is commonly seen in
human immunodeficiency virus positive individuals and AIDS patients. A model for oral candidiasis in
T-cell-deficient BALB/c and CBA/CaH nu/nu mice was established. After inoculation with 108Candida albicans
yeasts, these mice displayed increased levels of oral colonization compared to euthymic control mice and
developed a chronic oropharyngeal infection. Histopathological examination of nu/nu oral tissues revealed
extensive hyphae penetrating the epithelium, with polymorphonuclear leukocyte microabscess formation.
Adoptive transfer of either naive or immune lymphocytes into immunodeficient mice resulted in the recovery
of these animals from the oral infection. Reconstitution of immunodeficient mice with naive CD4?but not
CD8?T cells significantly decreased oral colonization compared to controls. Interleukin-12 and gamma
interferon were detected in the draining lymph nodes of immunodeficient mice following reconstitution with
naive lymphocytes. This study demonstrates the direct requirement for T lymphocytes in recovery from oral
candidiasis and suggests that this is associated with the production of cytokines by CD4?T helper cells.
Oral candidiasis is a predominant feature in patients suffer-
ing from human immunodeficiency virus (HIV) and AIDS (28,
35), and these patients, as well as those with chronic mucocu-
taneous candidiasis (27), commonly exhibit defects in cell-
The homozygous athymic nude mouse (nu/nu) is deficient in
T lymphocyte number and function, analogous to DiGeorge
syndrome in humans. In nu/nu mice, the thymus is almost
totally absent due to the failure of the development of the
thymic anlage, which arises from the ectoderm of the third
pharyngeal pouch (36). This leads to many defects in the im-
mune system, including depletion of lymphocytes from thymus-
dependent areas of lymph nodes and spleen, a much reduced
lymphocyte population composed mostly of B cells, relatively
normal immunoglobulin M (IgM) response to thymus-inde-
pendent antigens, very poor response to thymus-dependent
antigens, and greatly increased susceptibility to infection (37).
T-cell-deficient nude mice have been widely used to evaluate
the role of cell-mediated immune responses against Candida
albicans infections in systemic (12, 19, 22, 30, 39), vaginal (10,
11), and mucosal (gastrointestinal) (13, 14) models of the dis-
ease. Work by our group (C. S. Farah, T. Gotjamanos, and
R. B. Ashman, submitted for publication) has shown that an
oral infection of mild to moderate severity and relatively short
duration could be established in inbred mice of different ge-
netic backgrounds. However, although oral C. albicans infec-
tion is known to elicit cell-mediated immune responses, there
is no unequivocal evidence that T cells are essential for recov-
ery from a primary infection with this yeast. A model of oral
candidiasis in immunodeficient mice would allow immunolog-
ical manipulation of the system and facilitate evaluation of the
role of T cells in this infection.
As clinical observations support a role for T cells in oral
candidiasis, T-cell-deficient mice provide an ideal model to
study the role of T-cell-mediated immunity in oral candidiasis.
The present paper reports the establishment of an oral C.
albicans infection in T-cell-deficient mice and demonstrates
the critical role of T cells in the clearance of the yeast from the
oral tissues after primary infection.
MATERIALS AND METHODS
Mice. Specific-pathogen-free BALB/c and CBA/CaH female nude mice, their
heterozygous littermates, and euthymic controls, 6 to 8 weeks of age, were
purchased from the Animal Resources Centre, Perth, or from the Walter and
Eliza Hall Institute, Melbourne, Australia. These mice undergo routine micro-
biological screening and do not harbor C. albicans in the gut. Animal experi-
ments were approved by the Animal Experimentation Ethics Committee of the
University of Queensland and were carried out in accordance with the National
Health and Medical Research Council’s Australian Code of Practice for the Care
and Use of Animals for Scientific Purposes, 1997. Mice were housed in standard
cages and provided with food and water ad libitum.
Yeast. C. albicans isolate 3630, derived from a patient with cutaneous candi-
diasis, was obtained from the Mycology Reference Laboratory at the Royal
North Shore Hospital, Sydney, Australia, and stored at ?70°C in Sabouraud’s
broth with 15% (vol/vol) glycerol. For use, yeasts were grown in Sabouraud’s
broth for 48 h at room temperature with continuous agitation on a magnetic
stirrer. Blastospores were washed in phosphate-buffered saline (PBS) and ad-
justed to the appropriate concentration for inoculation.
Oral infection. Mice were inoculated orally with 108live C. albicans yeasts in
20 ?l of PBS. The infection was monitored by swabbing the oral cavity with
sterile cotton swabs moistened with sterile PBS and plating on Sabouraud’s agar
plates. Agar plates were incubated for 48 h at 37°C. All inoculation and sampling
procedures were conducted under halothane anesthesia using an inhalation
apparatus and a scavenging system. CFU were counted on Sabouraud’s agar
plates. The counts were assigned into five groups correlating with the level of
* Corresponding author. Mailing address: Oral Biology and Pathol-
ogy, The University of Queensland, Brisbane, QLD 4072, Australia.
Phone: 61 7 3365 8840. Fax: 61 7 3365 1109. E-mail: email@example.com
recoverable yeasts from the oral cavity. This provided a semiquantitative mea-
sure of the level of floridity of the infection. The scoring system used was as
follows: 0, no detectable yeasts; 1, 1 to 10 CFU/plate; 2, 11 to 100 CFU/plate; 3,
101 to 1,000 CFU/plate; and 4, 1,000? CFU/plate.
Histopathology. Mice were sacrificed at various time points throughout the
course of the experiment for histopathological examination of tissues. Thymus
glands were fixed in 10% neutral buffered formalin (pH 7.0), sectioned, and
stained with hematoxylin and eosin. Skulls were also fixed in buffered formalin
and decalcified in a 5% formic acid–sodium formate mixture. Frontal sections of
the skull were taken at approximately 3-mm intervals, with consecutive sections
stained with hematoxylin and eosin and according to the periodic acid-Schiff
technique. Sections were examined by light microscopy.
Footpad injection. Mice were injected subcutaneously in the footpad with 107
heat-killed C. albicans yeasts in 20 ?l of PBS. This dose has previously been
shown to produce optimal delayed-type hypersensitivity responses in mice (3).
The contralateral footpad was injected with an equal volume of sterile PBS.
Footpad thickness was measured using dial calipers (Mitutoyo). Each measure-
ment was repeated three times on a minimum of five mice.
Preparation of splenocytes. Spleens were removed from donor mice, and
spleen cells were obtained by pressing the spleens through a sterile metal sieve,
followed by filtration through an 80-?m nylon mesh. The cells were resuspended
in 6 ml of PBS, and lymphocytes were separated on a Ficoll gradient by under-
laying the cell suspension with 4 ml of Ficoll-Hypaque (Pharmacia Biotech,
Uppsala, Sweden). The gradient was centrifuged at 700 ? g for 25 to 30 min at
room temperature. The buffy coat interface was carefully removed, washed twice
in PBS, and resuspended in 1 ml of PBS. Viable lymphocytes were counted in a
hemacytometer after staining with trypan blue dye and adjusted to the appro-
priate concentration for injection into recipient mice.
Adoptive lymphocyte transfer in immunodeficient mice. At the height of oral
infection (day 6 following oral challenge), 3 ? 107nonimmune lymphocytes from
naive donor mice or 3 ? 107immune lymphocytes from previously immunized
donor animals were injected intravenously into the respective immunodeficient
recipient mice. Control animals received no cells and were used to monitor the
normal course of infection. Donor mice were primed orally with 108viable C.
albicans yeasts and rested for 4 to 8 weeks before use.
Thymus transplant. Immunodeficient mice were inoculated orally with 108
viable yeasts. At the height of infection (day 6), thymuses from naive inbred mice,
6 to 8 weeks of age, were surgically removed and transplanted into nude animals
subcutaneously under the right axilla. Control animals had sham operations
performed. The surgical sites were closed with wound clips. At the termination
of the experiment, the transplant site was examined visually and histologically to
determine whether grafting of the thymus was successful.
Mouse CD4?and CD8?T-cell enrichment. T-cell enrichment was carried out
using commercially available CD4?and CD8?mouse T-cell enrichment col-
umns (R&D, Minneapolis, Minn.), according to the manufacturer’s instructions.
Spleen lymphocytes from naive inbred mice were obtained as described above
and separated into CD4?and CD8?T cells on high-affinity negative-selection
columns. Briefly, 1 ml of monoclonal antibody cocktail was mixed with 2 ? 108
leukocytes in single-cell suspension and incubated at room temperature for 15
min. The cells were washed twice in 1? column buffer and resuspended in 2 ml
of the same buffer. The antibody-treated cells were then applied to a subset
column, incubated at room temperature for 10 min, and eluted with 10 ml of 1?
column buffer. The isolated cells were centrifuged at 200 ? g for 5 min and
suspended in PBS at the appropriate concentration for injection.
Fluorescence-activated cell sorting (FACS) analysis (CellQuest version 3.1,
FACSCalibur; Becton Dickinson, San Jose, Calif.) using fluorescein isothiocya-
nate (FITC)-conjugated rat anti-mouse CD4 (IgG2a) and R-phycoerythrin (PE)-
conjugated rat anti-mouse CD8 (IgG2a) antibodies (Pharmingen, San Diego,
Calif.) confirmed that the purity of cells isolated using these columns was 93.7%
for CD4 and 89.8% for CD8.
CD4?and CD8?T-cell reconstitution. BALB/c nude mice were infected with
108C. albicans yeasts. At the height of oral infection (day 6 following oral
challenge), nude recipient mice were reconstituted with naı ´ve CD4?or CD8?T
cells as follows: either 2.5 ? 106CD4?cells, 2.5 ? 106CD8?cells, or a
combination of 1.5 ? 106CD4?and 1.5 ? 106CD8?cells. A positive control
group received 3 ? 107naı ´ve lymphocytes, while a negative control group
received no cells.
Cell surface antigen staining. Spleens or lymph nodes were isolated as de-
scribed earlier and suspended in 0.1% PBS–NaN3. No Ficoll treatment was
required for lymph node cells. A total of 106cells were stained in 50 ?l of 0.1%
PBS–NaN3for 30 min at 4°C in the dark, using an appropriate concentration of
a fluorochrome-conjugated monoclonal antibody specific for a cell surface anti-
gen or isotype control: 1 ?l, 500 ?g of FITC-rat anti-mouse CD4 (IgG2a); 1.5 ?l,
200 ?g of R-PE-rat anti-mouse CD8 (IgG2a); 1 ?l, 500 ?g of FITC-rat IgG2a
isotype control immunoglobulin; and 1.5 ?l, 200 ?g of R-PE-rat IgG2a isotype
control immunoglobulin (Pharmingen, San Diego, Calif.) per ml. The cells were
washed twice in 0.1% PBS–NaN3at 350 ? g for 5 min and resuspended in 500
?l of 1% formalin. Cells were analyzed by FACS.
Cytokine ELISA. Lymphocytes isolated from the submandibular and superfi-
cial cervical lymph nodes were cultured ex vivo for 3 days at 4 ? 106cells/ml in
RPMI 1640 tissue culture medium without antigen stimulation. The supernatant
was collected, filtered through a 0.8-?m filter, and stored at ?20°C until ana-
lyzed. The culture supernatant was assayed for interleukin-4 (IL-4), IL-10, IL-12,
and gamma interferon (IFN-?) by enzyme-linked immunosorbent assay (ELISA)
using matched antibody pairs and recombinant cytokines as standards. Briefly,
Immuno-Polysorb microtiter plates (Nunc, Rothskild, Denmark) were coated
with capture rat monoclonal anti-IL-4 (IgG1), IL-10 (IgG1), IL-12 (IgG2a), or
IFN-? (IgG1) antibody (Pharmingen) at 1 ?g/ml in sodium bicarbonate buffer
overnight at 4°C. The wells were washed and then blocked with 1% bovine serum
albumin–PBS before the culture supernatants and the appropriate standard were
added to each well. Biotinylated rat monoclonal anti-IL-4, -IL-10, -IL-12, or
-IFN-? antibody (Pharmingen) at 2 ?g/ml was added as the second antibody.
Detection was carried out with streptavidin-peroxidase and tetramethylbenzi-
dine. The results were expressed as net Candida-induced counts, from which the
background was subtracted, and are shown as supernatant cytokine titer (pico-
grams per milliliter) (mean ? standard error of the mean [SEM]) from lympho-
cytes pooled from a minimum of three mice per group per time point.
RT-PCR. For reverse transcription-PCR (RT-PCR), total RNA was extracted
from oral tissues using Ultraspec RNA reagent (Biotecx Laboratories, Houston,
Tex.) according to the manufacturer’s instructions. cDNA was prepared by re-
verse transcription of 1 ?g of each RNA, using an oligo(dT)15primer and avian
myeloblastosis virus reverse transcriptase (Promega, Madison, Wis.). Briefly, 4 ?l
of 25 mM MgCl2solution, 2 ?l of 10? PCR amplification buffer [670 mM
Tris-HCl, 166 mM (NH4)2SO4, 4.5% Triton X-100, 2 mg of gelatin/ml] (BioTech
International, Nedlands, Australia), 2 ?l of a 10 mM deoxynucleoside triphos-
phate (dNTP) mix, 0.5 ?l of RNasin, 0.75 ?l (15 U) of avian myeloblastosis virus
reverse transcriptase, 0.5 ?g of oligo(dT)15primer, and 1 ?g of an mRNA sample
were incubated in a 20-?l reaction mix at 42°C for 1 h, heated to 99°C for 5 min,
then cooled on ice. cDNA was amplified by PCR in an amplification mix con-
sisting of 2 ?l of 25 mM MgCl2, 2.5 ?l of 10? reaction buffer, 2 ?l of 25 mM
dNTP mix, 0.5 U of Taq DNA polymerase (Geneworks, Thebarton, Australia),
the appropriate primer, and 1 ?l of cDNA in a total volume of 25 ?l. Negative
controls (without cDNA) were included for all primers used in each run.
The mixture was amplified using a PTC-100 programmable thermal cycler (MJ
Research Inc.). The amplification protocol was 94°C for 30 s, 60°C for 30 s, and
72°C for 1 min for 35 to 40 cycles. Following amplification, 10 ?l of product was
analyzed by electrophoresis through 2.5% (wt/vol) agarose gels. The gels were
stained with ethidium bromide and the bands were visualized with a UV trans-
illuminator (GelDoc 2000; Bio-Rad, Regents Park, Australia) with appropriate
software (MultiAnalyst version 1.1; Bio-Rad, Hercules, Calif.). Primer sequences
for CD4 and CD8 were obtained from published data (34) and synthesized by
Geneworks, Adelaide, South Australia. Data are representative of at least two
Statistics. Quantitative data were analyzed using the statistical features of
GraphPad Prism version 2.01 (GraphPad Inc., San Diego, Calif.). Student’s t test
and one-way analysis of variance were used, with a P value of ?0.05 considered
significant unless otherwise stated.
Colonization levels in nu/nu and nu/? mice. Athymic (nude)
mice bred on the BALB/c and CBA/CaH backgrounds were
infected orally with 108C. albicans yeasts and monitored for 21
days by swabbing the oral cavity. In both strains, higher num-
bers of yeasts were recovered from the oral cavities of these
animals compared to euthymic controls, and the severity and
the duration of the infection were significantly greater (P ?
0.001) (Fig. 1). The mice were chronically colonized, with the
infection lasting more than 70 days (data not shown). In the
BALB/c nu/? heterozygous mice, the infection was more se-
vere than that seen in the euthymic mice, but colonization
levels were only significantly different on day 8 for nu/? mice
VOL. 70, 2002 CD4?T CELLS IN ORAL CANDIDIASIS 725
(Fig. 2). The nu/? mice also cleared the infection within 21
Histopathology of oral tissues. Histopathological examina-
tion of the oral tissues from infected BALB/c (Fig. 3A) and
CBA/CaH nu/nu (Fig. 3B) mice showed hyphal elements pen-
etrating the keratinized epithelium and microabscess forma-
tion comprising mostly polymorphonuclear phagocytes (Fig.
3C). The two strains differed in the severity of the infection,
with an increased fungal burden seen in CBA/CaH nu/nu mice
(Fig. 3B). Sections of the tongue showed a markedly hyper-
plastic irregular keratinized epithelium, with almost complete
loss of surface papillae, acanthosis, and thickening and blunt-
ing of rete pegs. There was also a heavy surface hyperkeratosis.
The dorsal surface of the tongue carried an extensive and
heavy superficial infiltration of Candida cells. The tissues were
acanthotic and showed a loss of distinct stratum granulosum
and a consequent decrease in surface keratinization. The basal
half was loosely textured, with predominant intercellular
edema. There were multiple foci of transmigrating neutrophils
and a lesser number of superficial intraepithelial neutrophil
accumulations, consistent with a Munro’s microabscess (Fig.
The microabscesses were mostly confined to the superficial
zone of the epithelium in what might be described as a pseudo-
membrane (Fig. 3). The superficial lamina propria was fibrotic
and contained a moderately dense inflammatory infiltrate that
was predominantly lymphocytic/lymphohistiocytic, with scat-
tered neutrophils and occasional eosinophils. In CBA/CaH
nu/nu mice, the gingival tissues showed a heavily hyperplastic
keratinized epithelium. The sulcular and crestal gingival tissues
showed marked infection with candidal hyphae and associated
intraepithelial abscess formation consisting of clusters of neu-
trophils, with some neutrophils present in the superficial lam-
ina propria (data not shown).
Thymus transplantation. BALB/c nu/nu mice were trans-
planted with thymus glands from naive syngeneic mice 6 to 8
weeks of age. The thymus was transplanted into nude BALB/c
mice 6 days after colonization with C. albicans. Mice that
received the thymus transplant significantly reduced the infec-
tion by day 49, whereas control animals that only had sham
surgery performed were still heavily colonized with the yeast
Adoptive transfer of naive and immune lymphocytes.
BALB/c and CBA/CaH nu/nu mice were adoptively trans-
ferred with 3 ? 107lymphocytes from animals of the same
background. The lymphocytes were collected from either naive
or immune (orally primed) animals. Control animals received
no cells. BALB/c nude mice that received either naive or im-
mune lymphocytes cleared the infection by day 49, whereas the
control mice were still heavily colonized (P ? 0.001) (Fig. 5A).
CBA/CaH nude mice that received naive cells also significantly
reduced the Candida infection by day 49, whereas animals that
received immune cells were able to clear the infection by day
21 (Fig. 5B). A comparison of clearance rates following the
transfer of either naive or orally primed lymphocytes revealed
no significant difference in BALB/c nude mice, but showed
accelerated clearance in CBA/CaH mice that received immune
cells (Fig. 5).
Reconstitution with CD4?and CD8?T lymphocytes. Re-
constitution of infected BALB/c immunodeficient mice with
naive CD4?T cells resulted in a significant clearance of the
yeast from the oral cavity of these animals and eventual recov-
ery from chronic oral candidiasis (Fig. 6). The difference in
colonization levels was significant (P ? 0.001) from day 49
onwards. Animals reconstituted with CD4?T cells alone fol-
lowed a similar pattern of clearance to those reconstituted with
a pool of lymphocytes. CD8?T cells alone were unable to clear
FIG. 1. Time course of C. albicans clearance in BALB/c nu/nu
(A) and CBA/CaH nu/nu (B) immunodeficient mice compared to
wild-type controls following oral infection with 108C. albicans yeasts.
Bars represent scores (mean ? SEM) for a minimum of 30 mice (10
mice/group in three separate experiments). SEM is 0 if error bars are
not shown. All groups were significantly different (P ? 0.001) except
CBA/CaH nu/nu and CBA/CaH on day 1.
FIG. 2. Time course of C. albicans clearance in heterozygous
BALB/c nu/? and wild-type ?/? counterparts following oral infection
with 108C. albicans yeasts. Bars represent scores (mean ? SEM) for a
minimum of 10 mice/group. Each experiment was repeated twice. ?,
significantly different at P ? 0.05.
726FARAH ET AL.INFECT. IMMUN.
the infection in nu/nu mice, although there was a slight de-
crease in colonization levels compared to animals that received
no cells. At day 70, these animals still showed significantly
higher levels of colonization compared to positive control an-
imals reconstituted with lymphocytes (Fig. 6). Animals recon-
stituted with a mixed population of CD4?and CD8?T cells
were able to clear the infection in a fashion similar to that of
mice administered CD4?cells alone. Colonization levels were
significantly less than in controls (P ? 0.001) from day 49
onwards (data not shown).
Footpad swelling in immunodeficient mice after lymphocyte
reconstitution and thymus transplant. BALB/c mice reconsti-
tuted with immune lymphocytes showed greater footpad swell-
ing (P ? 0.001) than control mice that received no lymphocytes
and mice that received naive cells at 24, 48, and 96 h after
footpad injection (Table 1). Measurement of footpad swelling
in CBA/CaH mice also showed a difference between the im-
mune and the naı ´ve and control groups (Table 1). Footpad
swelling was significantly greater (P ? 0.001) in mice that
received immune lymphocytes than in those that received naive
cells. This situation existed for both BALB/c and CBA/CaH
mice at all time points following footpad injections (Table 1).
In thymus-transplanted mice, footpad swelling with heat-killed
Candida yeasts was significantly greater (P ? 0.05) than that
with PBS (data not shown).
There was a positive footpad response in mice reconstituted
with naive CD4?cells alone or a combination of CD4?and
CD8?T cells (data not shown). The footpad response seen in
mice reconstituted with naive CD8?cells alone was similar to
that in control animals (no lymphocyte transfer) and signifi-
cantly less (P ? 0.01) than that of CD4?-reconstituted mice
(data not shown).
Histopathology of oral tissues in nu/nu mice following lym-
phocyte reconstitution. Following lymphocyte reconstitution in
nude BALB/c mice, histopathological sections of the tongue
showed markedly fewer Candida hyphae penetrating the su-
perficial epithelium (Fig. 7A). The neutrophilic inflammatory
infiltrate was less obvious, but intraepithelial polymorphonu-
clear lymphocytes (PMNLs) were still found, and the lamina
propria was mildly lymphocytic (Fig. 7).
CD4?and CD8?T-cell expression in lymph nodes and
spleen after reconstitution. The majority of T cells detected in
lymph node and spleen of naive nude BALB/c mice were
phenotypically CD8?. Upon reconstitution of immunodefi-
cient mice with naive lymphocytes obtained from spleens of
syngeneic mice, the percentage of CD4?T cells rose to ap-
proximately 25% on day 4 and remained generally the same up
FIG. 3. Histopathological sections of tongue from immunodeficient BALB/c nu/nu (A) and CBA/CaH (B) nu/nu mice on day 6 after oral C.
albicans infection with 108yeasts. There is heavy infiltration of Candida hyphae penetrating the superficial hyperkeratotic epithelium, and large
numbers of PMNLs forming microabscesses (C). Sections were stained with periodic acid-Schiff. Bars, 50 ?m (A) and 100 ?m (B). Total
magnification, ?600 (C).
FIG. 4. Time course of C. albicans clearance in BALB/c nu/nu mice
after thymus transplantation. Bars represent scores (mean ? SEM) for
a minimum of 10 mice/group. Each experiment was repeated twice.
Comparison was made between animals that received a thymus trans-
plant (Thymus ?) and controls (Thymus ?) that received no thymus.
?, significantly different at P ? 0.001. SEM is 0 if error bars are not
VOL. 70, 2002 CD4?T CELLS IN ORAL CANDIDIASIS727
to day 21 after lymphocyte transfer (data not shown). The
CD8?subpopulation increased from about 4% on day 0 to
15% on day 4 and remained between 15 and 20% till day 21. A
CD4-to-CD8 ratio of 2:1 was achieved by day 4 after lympho-
cyte transfer in both spleen and draining lymph nodes. This
was sustained in the spleen, but decreased to approximately 1:1
in the submandibular and superficial cervical lymph nodes on
days 8 and 14 (data not shown).
CD4 and CD8 gene expression in oral tissues. Since CD4?
and CD8?T cells were capable of repopulating the draining
lymph nodes and spleen in nude mice after lymphocyte recon-
stitution, it was desirable to demonstrate the presence of mes-
sage for CD4 and CD8 in the oral tissues of these mice by
RT-PCR. Agarose gels stained with ethidium bromide re-
vealed no CD4 in oral tissues of nude mice infected with C.
albicans without lymphocyte reconstitution (Fig. 8, lane 3).
CD4 gene expression was detected in oral mucosa on days 4
and 8 after lymphocyte reconstitution (Fig. 8, lanes 5 and 7,
respectively). CD4 was not detected in the tongue until day 21
following lymphocyte transfer (data not shown). CD8 was de-
tected in infected controls (Fig. 8, lane 4) and reconstituted
animals at all time points examined (Fig. 8, lanes 6 and 8).
Cytokine production by lymph node cells by ELISA. ELISAs
conducted on culture supernatant of lymphocytes from the
draining lymph nodes of nude mice following lymphocyte re-
constitution showed that the overwhelming cytokine produced
was IL-12. This was apparent on days 8 (1,625 ? 262.1 pg/ml),
14 (1,059 ? 93.84 pg/ml), and 21 (1,405 ? 150 pg/ml) following
lymphocyte transfer. There was some IFN-? produced on day
8 (442.2 ? 68.93 pg/ml), but this soon returned to baseline
levels on days 14 (46.38 ? 4.37) and 21 (46.23 pg/ml). Only very
small amounts of IL-10 (38.9 to 67.7 pg/ml) were produced,
with no IL-4 detected in the cell supernatant at any time point.
The current study demonstrates that BALB/c and CBA/CaH
nu/nu mice are naturally susceptible to oral candidiasis and
provide an excellent model to study various aspects of suscep-
tibility and resistance to this disease. BALB/c nu/? mice were
slightly more susceptible to oral infection than their euthymic
congenic counterparts, but less susceptible than the immuno-
deficient nu/nu mice. A prolonged chronic oral infection could
be established in nu/nu mice which did not resolve for at least
70 days, confirming a requirement for T cells in recovery from
Previous work from our laboratory (21; Farah et al., submit-
ted), along with other studies of experimental C. albicans in-
fections of the mucosa (9, 17, 29, 33), has shown small numbers
of yeasts and hyphae colonizing the palate and tongue of nor-
mal (euthymic) mice. Histopathological examination of the
oral tissues from BALB/c and CBA/CaH nu/nu mice showed
abundant hyphal elements penetrating the epithelium and mi-
FIG. 5. Time course of C. albicans clearance in BALB/c nu/nu
(A) and CBA/CaH nu/nu (B) mice reconstituted with 3 ? 107immune
(orally primed) or naive lymphocytes. Bars represent scores (mean ?
SEM) for a minimum of 10 mice/group. Each experiment was repeated
twice. Comparison was made between animals that received lympho-
cytes and controls that received no cells. ?, significantly different at P ?
0.001. SEM is 0 if error bars are not shown.
FIG. 6. Time course of C. albicans clearance in BALB/c nu/nu mice
reconstituted with 2.5 ? 106naive CD4?or CD8?T cells. The positive
control group received 3 ? 107naive lymphocytes, while the negative
control group received no cells. Bars represent scores (mean ? SEM)
for a minimum of 10 mice/group. Each experiment was repeated twice.
Comparison was made between animals that received either CD4?or
CD8?T cells and negative controls. ?, significantly different at P ?
0.001 for CD4 group; F, significantly different at P ? 0.05 for CD8
group. Positive control group was significantly different from negative
control group at P ? 0.001 from days 21 to 70.
728 FARAH ET AL.INFECT. IMMUN.
croabscess formation, consisting mostly of polymorphonuclear
phagocytes. The pseudomembrane-like appearance of C. albi-
cans lining the oral cavity in nu/nu mice, especially CBA/CaH,
is very similar to that in clinical thrush observed in patients
with HIV and AIDS (28, 35). The increased fungal burden in
the oral cavity of the CBA/CaH nu/nu strain might be related
to the genetic susceptibility of the CBA/CaH strain to both oral
(Farah et al., submitted) and systemic colonization (1–3, 5, 6).
The chronic oral infection established in nude mice provided
a system in which immunological manipulation was exploited
to confirm the importance of T cells in this infection. Nude
mice that received a thymus transplant cleared the infection, as
did mice that received syngeneic lymphocytes, confirming a
role for T cells and thymus-dependent immunity in this model
of oral infection. However, despite the difference in the sever-
ity of oral colonization, there was no difference in the rate of
clearance between BALB/c nu/nu and CBA/CaH nu/nu mice
after the transfer of naive lymphocytes into these mice. This is
consistent with previous observations that tissue susceptibility
to C. albicans is determined by innate and not by adaptive
immune responses (7).
Surprisingly, lymphocytes transferred from orally primed an-
imals markedly accelerated clearance in the CBA/CaH nude
mice, but were no more effective than naive cells in promoting
clearance of the yeast from the oral cavity of BALB/c nu/nu
mice. This result is consistent with the increased antibody-
mediated resistance against systemic challenge displayed by
immune CBA/CaH compared to BALB/c mice (4, 8). The basis
for this difference is unknown at present; however, BALB/c
mice appear to have stronger innate immune defenses than
CBA/CaH mice, so that the window for immunological en-
hancement of resistance is smaller in this strain. Since the host
response is most probably mediated by cytokines produced by
CD4?T cells, it is possible that after activation, cells from
naive mice can deliver levels of cytokines that are sufficient for
optimal or near optimal recruitment and/or activation of the
phagocytic effector cells. If this is the case, the increased num-
ber of Candida-specific cells present in the immune cell pop-
ulation may only be able to make a small difference to the
magnitude or rapidity of the host response, particularly in the
There was a strong expression of delayed-type hypersensi-
tivity in both BALB/c and CBA/CaH nude mice following the
adoptive transfer of lymphocytes from primed but not naive
mice. This is in sharp contrast to the patterns of clearance of
oral infection after adoptive transfer of immune and naive cells
FIG. 7. Histopathological section of tongue from immunodeficient BALB/c nu/nu mouse following lymphocyte reconstitution (A) (day 35
posttransfer), and infected control (B) that received no cells. Mice were infected with 108C. albicans yeasts and reconstituted with 3 ? 107naive
lymphocytes on day 6 postinfection. Sections show fewer hyphae penetrating the epithelium in the reconstituted mouse, with some intraepithelial
PMNLs (A), compared to the heavy hyphal penetration and PMNL microabscess formation in the untreated control (B). Sections were stained
with periodic acid-Schiff. Bars, 45 ?m.
TABLE 1. Footpad swelling following lymphocyte transfer in BALB/c and CBA/CaH nu/nu mice infected with C. albicansa
Mean (SEM) increase in footpad thickness, mm
BALB/c miceCBA/CaH mice
ImmuneNaive Control ImmuneNaiveControl
aResults are presented as mean (SEM) increase in footpad thickness following injection with 107heat-killed C. albicans yeasts in a minimum of five mice that were
reconstituted with either immune or naive lymphocytes. Each experiment was repeated twice. Comparison was made between animals that received lymphocytes and
controls that received no cells, and results were significantly different at P ? 0.001 only for animals that received immune cells compared to controls at all time points.
VOL. 70, 2002 CD4?T CELLS IN ORAL CANDIDIASIS729
discussed above, but is consistent with the reported lack of
correlation between the magnitude of delayed-type hypersen-
sitivity responses and clearance of systemic candidiasis (23).
B-cell epitopes are known to differ between mouse strains (18),
and it is likewise possible that subsets of CD4?T cells also
recognize different antigenic peptides of C. albicans presented
in the context of major histocompatibility complex class II.
Thus, delayed-type hypersensitivity cells may represent a mem-
ory subpopulation of CD4?cells whose functions are irrele-
vant to the recruitment and/or activation of the effector cells
responsible for clearance of the yeast from the oral cavity.
Naive CD4?and CD8?T-cell population subsets separated
by a negative selection protocol were introduced intravenously
into BALB/c nu/nu mice to examine the contribution of each to
recovery from oral infection. The results show that CD4?but
not CD8?T cells are capable of reducing the fungal burden in
the oral tissues and eventually lead to the recovery of these
mice from chronic oral candidiasis. Animals reconstituted with
either CD4?cells alone or a mixture of CD4?and CD8?T
cells exhibit the same pattern of clearance as animals recon-
stituted with a pool of naive mixed lymphocytes. A significant
delayed-type hypersensitivity response was also observed fol-
lowing reconstitution with CD4?but not CD8?T cells. It was
clear that CD4?T cells were able to localize in the infected
oral mucosa and exert their activity there, as evidenced by the
CD4 gene expression in the oral tissues following reconstitution.
Although animals that received only naive CD8?T cells did
not clear the infection, there was a slight decrease in coloni-
zation levels, which might have been due to the carryover of
CD4?T cells in the transferred cells. CD4?cells constituted
only a small number of the T-cell population in the lymph
nodes of mice reconstituted with CD8?T cells; and control
mice that received no cells displayed comparable numbers of
CD4?T cells to that of the CD8?group (data not shown).
T-cell numbers in nude mice tend to increase with age (32),
and this might account for the slight decline in yeast CFU recov-
ered from these control mice late in the course of infection.
While ?/? T cells often predominate in oral mucosal epithe-
lium, the percentages of ?/? T cells are always considerably
higher than in the periphery (24, 31, 38). ?/? T cells show a
sharp increase in number following either oral (17, 20) or
gastrointestinal (25) infection with C. albicans, and in a recent
study, mice made deficient in ?/? or ?/? T cells (?- and ?-chain
T-cell receptor knockout mice, respectively) were shown to be
more susceptible to orogastric candidiasis than immunocom-
petent controls (26).
The most abundant cytokine detected in the submandibular
and superficial cervical lymph nodes of infected BALB/c nude
mice following reconstitution with naive lymphocytes was IL-
12. IL-12 is produced by phagocytic cells, B cells, and dendritic
cells and acts on T lymphocytes and NK cells by inducing
proliferation and production of cytokines, especially IFN-?. In
vivo, IL-12 has been shown to be required for the induction of
Th1 responses in systemic candidiasis (41), although its action
may also be regulated by IFN-? (15). In a model of gastroin-
testinal candidiasis, both Th1 cytokines, such as IFN-?, and the
Th2 cytokines IL-4 and IL-5 were produced by CD4?cells
from Peyer’s patches and mesenteric lymph nodes at a time
when the fungus was cleared from the stomach and intestine
(16). In addition, treatment with soluble IL-4 receptor, which
increased Th1 cells, was associated with enhanced yeast clear-
ance. It was concluded that activation of Th1- but not Th2-like
responses may be responsible for controlling gastrointestinal
candidiasis and generating protective immunity. In contrast to
these results, others have reported that neither IL-2 nor IFN-?
played a role in protecting the oral cavity from candidal infec-
tion (14), even though CD4?T lymphocytes were essential for
From the data presented in the current study and those
reported by others (14–16), there is little evidence that IL-2,
IL-4, and IL-10 play a significant role in mucosal candidiasis.
IL-12 and IFN-? were found in recovering reconstituted mice
and may play some role in the clearance of C. albicans yeasts
from the oral tissues of these animals. They are known to be
involved in natural and acquired aspects of the immune re-
sponse, and it is perhaps some aspect of this interaction that
underlies the effector function of CD4?T cells in the host
response against oral candidiasis.
In conclusion, the present study demonstrates the essential
role of CD4?T cells in the clearance and recovery of immu-
nodeficient mice from chronic oral candidiasis. Although these
data are consistent with the body of evidence that implicates
Th1 cytokines in recovery from candidiasis (40), the precise
role of these cytokines in recovery from oral candidiasis re-
mains to be determined.
This work was supported by the National Health and Medical Re-
search Council of Australia and the Australian Dental Research Foun-
dation. C.S.F. was supported by an NH&MRC Dental Postgraduate
We thank Slavica Pervan for preparation of the histological samples.
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Editor: T. R. Kozel
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