INFECTION AND IMMUNITY, Dec. 1994, p. 5353-53600019-9567/94/$04.00+0Copyright X 1994, American Society for Microbiology

A Mannoprotein Constituent of Candida albicans That ElicitsDifferent Levels of Delayed-Type Hypersensitivity, Cytokine

Production, and Anticandidal Protection in MiceANTONELLA MENCACCI,1 ANTONELLA TOROSANTUCCI,2 ROBERTA SPACCAPELO,1

LUIGINA ROMANI,1 FRANCESCO BISTONI,' AND ANTONIO CASSONE2*

Microbiology Section, Department of Experimental Medicine and Biochemical Sciences, University of Perugia, Perugia,'and Laboratory of Bacteriology and Medical Mycology, Istituto Superiore di Sanita, Rome,2 Italy

Received 19 May 1994/Returned for modification 7 July 1994/Accepted 3 October 1994

To identify major immunogenic constituents of Candida albicans, the effect of a mannoprotein fraction(MP-F2) on the elicitation of a delayed-type hypersensitivity (DTH) reaction, cytokine production, andprotection from a virulent Candida challenge in a mouse candidiasis model was studied. In mice immunizedwith whole cells of a low-virulence strain of C. albicans and thus protected against a challenge with a highlyvirulent strain of this fungus, MP-F2 was able to elicit a strong DTH response that was accompanied bysplenocyte proliferation in vitro in the presence of Candida antigen. The supernatants of MP-F2-stimulatedsplenocyte cultures contained gamma interferon (IFN-,y, a typical CD4+ T helper-1 (Thl) cytokine, but no

interleukin-4, (IL-4), a typical CD4+ Th2 cytokine. IFN--y was produced by CD4+ cells, and its level could begreatly increased by the addition of anti-IL-4 or, mostly, anti-IL-10 antibodies to the CD4+ cell cultures. Upona suitable schedule of immunization, MP-F2 was also able to induce a vigorous DTH response inCandida-uninfected mice, a response that could be efficiently transferred into naive recipients by CD4+ cellsfrom the spleens of MP-F2-immunized mice. The immunization described above also conferred to mice a lowdegree of protection against a virulent Candida challenge, both in terms of median survival time and in thenumber of Candida cells in the kidney. However, while DTH induction by MP-F2 was as strong as that inducedby whole cells, MP-F2-induced protection was significantly weaker than that conferred by Candida whole-cellimmunization. Mice immunized with either MP-F2 or Candida whole cells had an inverted ratio between thenumber of CD4+ splenocytes producing IFN-,y and that of cells producing IL-4, compared with nonimmunizedanimals. However, the number of IL-4-producing CD4+ cells was significantly higher in MP-2-vaccinated,weakly protected mice than in Candida whole-cell-vaccinated, highly protected animals. Overall, our data suggestthat the MP-F2 fraction contains one or more major immunogens of C. albicans which are capable of interfering withthe balance of CD4+ Thl and Th2 responses that is so critical in the outcome of host-Candida relationship and are

thus potentially relevant in the mechanisms of Candida-specific DTH regulation and protection.

The yeast Candida albicans is one of the leading causes ofdisseminated fungal disease, in which impairment of the hostimmune system is one major factor predisposing to infection.Both natural (6, 8, 18) and adaptive (1, 15, 34) immuneresponses have been shown to contribute extensively to thehost's resistance to candidiasis. We have recently reported thatselective activation of either one of the CD4+ T helper (Th)lymphocyte subsets occurs in mice infected with C. albicans(37, 41). A strong correlation has been found between diseaseoutcome and the nature of the predominant Th-cell response,with healer mice developing a Thl-type response (32, 39) andnonhealer mice developing a predominant Th2 response (39,42). Both host genetics and C. albicans strain factors werefound to contribute to the generation of the anticandidal Thphenotypes. Thus, in genetically resistant mice, a protectiveThl response to virulent C. albicans was observed after mousevaccination with a live, low-virulence strain of the fungus;however, the same attenuated vaccine resulted in nonprotec-tive Th2 responses in genetically susceptible mice (37). Alter-ation of the disease outcome and Th conversion were obtainedin healer and nonhealer mice by changing their immune status

* Corresponding author. Mailing address: Laboratory of Bacteriol-ogy and Medical Mycology, Istituto Superiore di Sanita, Viale ReginaElena, 299, 00161 Rome, Italy. Phone: 0039-06-4440014. Fax: 0039-06-491723.

at the time of infection by means of specific cytokine neutral-ization (32, 35, 39, 42).The importance of the strain of C. albicans in triggering the

predominant Th subset is probably reflected in the kind ofantigens expressed by the fungal cells. In this line, cell wallcomponents of C. albicans are known to elicit or modulate a

variety of immune responses both in humans (4, 11, 21, 29) andin experimental settings (11, 19, 20). In particular, a manno-

protein (MP)-rich extract was found to contain componentsendowed with the ability to act as recall antigens in inducinglymphoproliferation (4, 33), production of cytokines, such as

gamma interferon (IFN--y), interleukin-2 (IL-2), IL-1p, tumornecrosis factor alpha, IL-6, and granulocyte-monocyte colony-stimulating factor (4, 5, 46), as well as non-major histocompat-ibility complex-restricted, cell-mediated cytotoxicity in culturesof peripheral blood mononuclear cells (PBMC) from healthyhuman donors (3). Interestingly, such activities were found tobe deeply depressed in individuals highly susceptible to oppor-

tunistic candidal infections, such as human immunodeficiencyvirus-infected patients (33) and cancer patients (2). In mice,candidal MP strongly activates natural immunity effectors suchas natural killer cells, macrophages, and polymorphonuclearcells (14, 29, 43). Thus, an immunoadjuvant effect was seen intumor-bearing mice treated with cell wall fractions from C.albicans (12).

Considering the immunoregulatory role exerted by T lym-

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phocytes in the generation of anticandidal responses (15, 36,37), it was of interest to evaluate the ability of MP componentto elicit or detect specific Th functions in mice infected with C.albicans. For this purpose, one major MP fraction of MP-richextract (MP-F2), which has been recently reported to containan immunodominant 65-kDa MP (45), was studied. It wasfound that MP-F2 was able to elicit a strong delayed-typehypersensitivity (DTH) reaction in mice vaccinated with low-virulence C. albicans cells. Candida-specific DTH reactivitycould also be induced in naive mice by immunization withMP-F2. This reactivity could be transferred upon naive recip-ients by means of IFN--y-producing, CD4+ splenocytes fromMP-F2-treated mice. Finally, strong DTH induction was asso-ciated with a low degree of protection from challenge with avirulent strain of C. albicans.

MATERIALS AND METHODS

Mice. Hybrid (BALB/cCr x DBA/2Cr)Fl (CD2F1 [H-2dIH-2d]) mice were obtained from Charles River Breeding Labo-ratories, Calco, Milan, Italy. Both male and female mice,ranging in age from 2 to 4 months, were used.

Yeasts and infections. The origin and characteristics of thetwo strains of C. albicans (CA-6 and PCA-2) used in this studyhave already been described elsewhere in detail (15, 37).Briefly, the highly pathogenic strain CA-6 was isolated from aclinical specimen; the agerminative low-virulence strain PCA-2(used as a C. albicans vaccine) is an echinocandin-resistantmutant of the parent strain 3153A. All yeasts were grown tothe stationary phase at 28°C under slight agitation in low-glucose Winge medium composed of 0.2% (wt/vol) glucose and0.3% (wt/vol) yeast extract (BBL Microbiology Systems, Cock-eysville, Md.). For infection, yeast cells were harvested bylow-speed centrifugation (1,000 x g), washed twice in phos-phate-buffered saline, and diluted to the desired density to beinjected intravenously (i.v.) into mice in a volume of 500 RIl.Quantification of yeast cells in the kidneys of infected mice wasperformed by a plate dilution method using Sabouraud dex-trose agar, and the results (means ± standard errors of themeans) were expressed as the number of CFU per organ.

Preparation of MP extracts of C. albicans. A crude MPextract was obtained from washed-yeast-form C. albicans BPserotype A as previously reported (4, 11, 43, 46). MP-F2, onemajor MP fraction of MP extract, was subsequently separatedby ion-exchange chromatography on a DEAE Sephadex A-50column, as previously described (46). Only one defined batchof MP-F2 was used for all experiments. This batch (identifiedas MP-F2-16) contained 92.5% mannan and 6.4% protein,which were assayed as reported elsewhere (45). The prepara-tion was passed through a Sepharose-polymyxin B column(Sigma) to ensure decontamination from bacterial endotoxin(29). A full description of the basic chemistry and molecularcomposition of MP-F2 fraction has been previously published(45, 46).

In vivo treatment with the MP fractions. MP-F2 was dis-solved in saline and injected intraperitoneally (i.p.) or subcu-taneously (s.c.) at 10 and 30 ,ug per mouse on days 16, 13, and10 before i.v. injection of 2 x 105 CA-6 cells. A booster dose,which was three times higher than those injected previously,was given 4 days before infection. Controls included parallelgroups of mice injected either with vehicle alone or with anMP-rich fraction, which was prepared from Saccharomycescerevisiae by the same procedure as that used for the MP-F2material and contained roughly the same amounts of polysac-charide and protein as MP-F2. This S. cerevisiae fraction wasreferred to as the irrelevant fraction (IF).

DTH assay. A direct assay system for measuring the DTHresponse to cell surface antigens was used (15, 37), in which 2x 10' whole C. albicans heat-inactivated cells (H-CA cells), theimmunoactive fraction MP-F2 (10 or 1 ,ug), or the IF (10 ,ug)was inoculated into the footpads of C. albicans-infected mice ina volume of 40 RI. The DTH reaction was recorded 24 h laterby weighing the footpads as a measure of swelling, and theresults were expressed as the increase in the weight of the righthind footpad over that of the saline-injected left hind counter-part. Data were expressed as the means ± standard errors ofsix to eight mice per group. In the adoptive transfer of DTHreactivity, prospective recipients of the footpad challenge wereinjected i.v. 24 h earlier with 107 CD4+ lymphocytes fromdonors that had been pretreated with MP-F2 or IF andinjected with CA-6, and harvested 6 days after challenge.

Splenocyte proliferation assay. Unfractionated spleen cells(5 x 106/ml), suspended in RPMI 1640 medium supplementedwith 5% fetal calf serum and 5 x 10-5 M 2-mercaptoethanol,which is referred to as complete medium, were cultured in96-well round-bottom microtiter plates without stimuli or with1 or 0.1 pLg of concanavalin A (ConA [Sigma Chemical Co., St.Louis, Mo.]) per ml or 5 x 105 or 5 x 104 H-CA cells to obtaincell-to-cell ratios of 10:1 and 100:1 or different concentrationsof MP-F2 or IF. The plates were incubated in a 10% CO2incubator at 37°C and were harvested after 72 h of culture.Eight hours before harvesting, radiolabeled thymidine (Amer-sham International, Amersham, United Kingdom) was addedat a final concentration of 0.5 ,uCi per well. Results wereexpressed as counts per minutes ± standard errors aftersubtraction of background control values, i.e., counts perminute from the splenocyte culture without stimulant.

Production of culture supernatants containing cytokineactivity. Unfractionated spleen cells (5 x 106/ml) suspended incomplete medium were cultured with H-CA (at a cell-to-cellratio of 100:1) or with MP-F2, IF, or ConA at 10 ,ug/ml each.In some experiments, CD4+ cells, positively selected frompools of spleen cells by means of a panning procedure usinganti-murine CD4 monoclonal antibody (MAb) GK1.5 (39, 41),were cultured for 48 h with H-CA or ConA or MP-F2 in thepresence or absence of anti-IL-4 or anti-IL-10 MAb (at 10,ug/ml each) (17). After incubation at 37°C for 48 h, culturesupernatants were harvested by centrifugation and were as-sayed for cytokine contents.

Cytokine assays. Culture supernatants were analyzed forIFN--y and IL-4 contents by two-site enzyme-linked immu-nosorbent assay as described previously (15, 37, 39, 41). ForIFN-y assays, the plates were coated with the rat-anti-murineIFN-y MAb R4-6A2 as the capture reagent, and biotinylatedMAb AN-18.17.24 was used in combination with streptavidin-horseradish peroxidase. IL-4 content was determined by usingMAb llBil as the coat and NIP-conjugated BVD6-24G2(kindly provided by Robert Coffman, DNAX Research Insti-tute, Palo Alto, Calif.) as the second step. Standard curves,which were constructed with known amounts of recombinantIFN--y and IL-4, were used for reference in calculation ofresults (means ± standard errors), which were expressed asnanograms per milliliter.Enumeration of CD4+ cells producing IFN-y, IL-4, and

IL-10. CD4+ lymphocytes (>95% pure) were positively se-lected from pools of spleen cells as described above. Theprocedure for enumeration of cytokine-secreting individualCD4+ cells was based on an ELISPOT assay, as describedelsewhere (7, 38). Briefly, 96-well nitrocellulose membranefiltration plates (Millititre HA; Millipore Corp., Bedford,Mass.) were precoated overnight with the anti-IFN--y MAbR4-6A2, the anti-IL-4 MAb llBl1, or the anti-IL-10 MAb

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Antigen Dose Days Footpad weight increase (mg)7.5 1s 22.6 30

Saline - 14

H-CA 2 x 10 cells 7- 14

28

MP-F2 10pg_14

- " 28

MP-F2 Ie 7114285

IF 10l g 71428

FIG. 1. Elicitation of the DTH reaction by whole C. albicans or itsimmunoactive fraction, MP-F2, in CD2F1 mice that had been chal-lenged with C. albicans cells a number of days previously.

SXC-2, and CD4+ cells were then incubated at 102 to 104 cellsper well for 20 h at 37°C in a 7% CO2 incubator. The secondaryantibodies were biotinylated MAb AN-18.17.24 for IFN--y, NIPconjugate BVD6-24G2 for IL-4, and biotinylated MAb SXC-1for IL-10, the enzyme was avidin-alkaline phosphatase conju-gate (Vector Laboratories, Burlingame, Calif.), and the sub-strate was 5-bromo-4-chloro-3-indolylphosphate-p-toluidinesalt, which was used in combination with the catalyst nitrobluetetrazolium chloride (BRL Life Technologies, Gaithersburg,Md.). Spots were counted with the aid of a dissecting micro-scope, and results were expressed as the number of cytokine-producing CD4+ lymphocytes per 105 cells calculated usingreplicates of serial twofold dilutions of cells pooled from fivemice per group.

Statistical analysis. Student's t test was used to determinestatistical significance between controls and treatment groups.Each experiment was repeated three to five times. In vivogroups consisted of six to eight animals. The data reported arefrom one representative experiment.

RESULTS

MP-F2 elicits DTH reactivity in C. albicans-infected mice.Because development of DTH is strictly associated with theactivation of CD4+ Thl cells (15, 37, 39, 41), we investigatedthe ability of MP-F2 to elicit DTH reaction in CD2F1 miceinjected i.v. with the low-virulence C. albicans cells, i.e., PCA-2.Previous studies demonstrated the onset of protective Thl-mediated anticandidal immunity in mice injected with PCA-2(32, 35, 37, 39, 42). At 7, 14, and 28 days after PCA-2 infection,different doses of MP-F2 were injected into the footpads of themice. Irrelevant MP fractions from S. cerevisiae and inactivatedC. albicans cells served as negative and positive controls,respectively. Twenty-four hours later, the increases in footpadweights were measured. The results (Fig. 1) showed thatMP-F2 was able to elicit strong, dose-dependent DTH reactiv-ity. When 10 ,ug of MP-F2 was employed, the extent and timeof appearance of the DTH reaction were comparable to thoseobtained with inactivated C. albicans cells. No such reactivitywas obtained with the IF.MP-F2 induces proliferation of splenocytes from C. albicans-

infected mice. MP-F2 was found to behave as a recall antigenin inducing proliferation and cytotoxicity in PBMC fromhealthy, Candida-sensitized individuals (4). We examined theability of MP-F2 to induce lymphoproliferation in vitro bysplenocytes from PCA-2-infected CD2F1 mice, using differentconcentrations of the fraction. Comparisons were made withthe proliferative responses induced by stimulation with themitogen ConA, inactivated C. albicans cells, and the IF. The

Es6

7 14 20

12 -

v-100 14

7 14 28 Days

FIG. 2. Proliferation of splenocytes from CD2F1 mice infectedwith C. albicans a number of days previously and stimulated in vitrowith 10 (-) or 0.1 (l) ,ug of ConA, 5 x 105 (a) or 5 x 104 (El) H-CAcells, MP-F2 at 10 (-) or 1 (El) j,g/ml, or IF at 10 (a) or 1 (E) j,g/ml.Control values, i.e., counts per minute of splenocytes cultured alone,never exceeded 800 cpm and were always subtracted.

results are shown in Fig. 2. The proliferation obtained inresponse to ConA was constantly present throughout theinfection period. Similarly, the MP-F2 fraction was able toinduce a proliferative response that was detectable at any timepoint assayed. Interestingly, the response to the fraction waseven higher than that elicited with whole C. albicans cells 2weeks postinfection. Minimal or no proliferative response wasobtained with the IF. No proliferation was obtained by cultur-ing splenocytes from naive, uninfected mice with MP-F2 (datanot shown).MP-F2 stimulates cytokine production by splenocytes from

infected mice. Splenocytes from mice vaccinated with PCA-2produce a variety of cytokines in vitro upon restimulation withthe C. albicans cells or ConA (32, 35, 37, 39, 42). We assessedthe potential of MP-F2 to stimulate cytokine release bydetermining the cytokine contents of supernatants from cul-tures of MP-F2-stimulated splenocytes from C. albicans-in-fected mice. The levels of cytokines released were comparedwith those found in culture supernatants of the cells stimulatedwith ConA, C. albicans cells, or the IF. Overall, the pattern ofcytokines produced in response to MP-F2 was not dissimilar tothat obtained in response to C. albicans cells as antigen and thepolyclonal stimulator ConA, as illustrated in Fig. 3. IFN--y wasproduced to about the same extent at any time point afterinfection, while no IL-4 could be detected after stimulationwith either C. albicans cells or MP-F2. Very low levels of IL-4were detected in polyclonal-stimulated splenocytes early dur-ing C. albicans infection (Fig. 3). No IL-10 was detected inMP-F2-stimulated cultures (data not shown).

Because one major source of IFN--y in murine candidiasis isthe CD4+ Thl subset (40), it was of interest to determine theability of CD4+ cells from PCA-2-infected mice to releaseIFN--y in response to MP-F2. To this purpose, CD4+ lympho-cytes, which were positively selected from splenocytes of miceinjected with C. albicans 14 days previously, were stimulated invitro with MP-F2 in the presence of irradiated histocompatiblesplenocytes as antigen-presenting cells. The results (Fig. 4)indicated that the level of IFN-y produced by CD4+ cells uponstimulation with MP-F2 was higher than that found in unstimu-lated cultures and lower than that obtained in the presence of

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so -

40 -

7e 30-

20 -

10-

5 -

4 -

3-

|LI Con A E MP-F2H-CA IF

lIo1

r-M-.

0 7 14 28 0 7 14 28 0 7 14 28 0 7 14 28Days

0 7 14 28 0 7 14 28 0 7 14 28 0 7 14 28Days

FIG. 3. Cytokine production of splenocytes of CD2F1 mice injected with C. albicans a number of days previously and cultured in vitro withConA (10 pLg/ml), H-CA (5 x 104 cells), or MP-F2 and IF at 10 p.g/ml each. Cytokine levels in unstimulated splenocytes were below the detectionlimits of the assay. 0, cultures of naive splenocytes. *, not detectable (below the detection limits of the assay).

whole inactivated yeast cells. Because the presence of even

very low amounts of IL-4 and IL-10 could adversely affectIFN--y production in vitro (22, 30), in selected experiments thecultures were added to either anti-IL-4 or anti-IL-10 MAbs. Inthese cultures, the MP-F2-induced IFN--y production could beremarkably increased, reaching a level close to that obtainedafter stimulation with ConA, by neutralization of IL-4 and,particularly, IL-10 (Fig. 4). The effect on anti-IL-10 MAb infostering IFN--y production was also detected in unfraction-ated spleen cells of C. albicans-immunized animals (seeabove). In one typical experiment, IFN--y production by MP-F2-stimulated splenocytes was 6.0 ± 0.5 and 8.8 ± 0.4 ng/ml inthe presence of anti-IL-4 and anti-IL-10 MAbs, respectively,compared with 5.4 0.8 ng of IFN--y per ml in the absence ofanti-cytokine antibody. Overall, the above data indicate that (i)the MP-F2 fraction is able to induce activation of the CD4+Thl cells and (ii) this activation is largely responsible for IFN--yproduction by the splenocytes of C. albicans-immunized ani-mals.

Effect of immunization with MP-F2 on the course of C.albicans infection. The finding that the MP-F2 fraction stronglyinduced the production of Thl cytokines in vitro prompted us

to evaluate its effect on the course of systemic infection withvirulent C. albicans cells. To this purpose, different doses of the

Stimulation with: IFNy (ng/ml)2 4 6 a 10

None _

Con A _

H-CA

MP-F2

' + Mabot IL-4

' + MabaIL-10e

FIG. 4. MP-F2-induced IFN--y production in vitro by CD4+ spleno-cytes from mice that had been infected with PCA-2 14 days previously.Cells were cultured with 10 ,ug of MP-F2 per ml for 48 h in thepresence or in the absence of 10 ,ug of either anti (ot)-Il-4 or anti(ox)-IL-10 MAb per ml. ConA and H-CA were used at 10 ,ug/ml and 5X 104 cells per ml, respectively.

MP fraction were administered by different routes into CD2F1mice a number of days before the infectious challenge withCA-6 cells; then, mice were monitored for mortality, numberof yeast cells in the kidneys, development of DTH reactivityand number of cytokine-producing CD4+ cells. Table 1 showsthe results of one representative experiment of five that were

performed with similar results. A significant increase in mediansurvival time was achieved by pretreating mice, four timesbefore infection, with 10 p.g of MP-F2 given i.p., but not s.c.Nevertheless, the protective effect obtained could not beincreased by using higher doses of the fraction (such as 50 and100 ,ug) or the use of the intrasplenic deposition route (datanot shown). Protection was associated both with the presenceof a lower number of yeast cells in the kidneys, compared withsaline-pretreated controls, and with the onset of DTH reactionto C. albicans cells. This DTH reaction was comparable to thatinduced by vaccination with PCA-2 cells, which was, however,much more protective (especially in terms of overall mortality)than immunization with the MP-F2 fraction (Table 1). Itshould be noted that CD4+ cells from the spleens of MP-F2-immunized (10 pRg) mice could transfer DTH reactivity uponnaive recipients (the increase in footpad weight was 7.3 ± 0.4mg compared with the 3.1 ± 0.6 mg of weight increase in thefootpads of mice receiving CD4+ cells from naive, uninfectedmice), thus indicating that MP-F2 participates in both thesensitization and the effector phases of DTH reactivity. Pre-treatment of mice with the IF did not result in any of the effectsobtained with MP-F2. Altogether, these results suggest thatthe MP-F2 fraction could exert a weak anti-C. albicans protec-tive effect in mice which is accompanied by, but not quantita-tively correlated with, the generation of strong, C. albicans-specific DTH.

Effect of immunization with MP-F2 on the numbers ofIFN--y-, IL-4-, and IL-10-producing CD4+ cells. Since theoutcome of infection with C. albicans depends on the type ofTh subset activated (41), we determined whether the resultsdescribed above could be associated with changes in the type ofT helper cells activated. Thus, the numbers of CD4+ spleno-cytes producing IFN-y, IL-4, and IL-10 after infection withvirulent C. albicans CA-6 cells were calculated by means of an

ELISPOT assay. Fresh CD4+ lymphocytes were obtained fromnonpretreated or MP-F2-, IF-, or PCA-2-pretreated CD2F1

0 7 14 28 0 7 14 28 0 7 14 28 0 7 14 28Days

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c 0

=

00

.s

2

d

z

a 110-

. 90

70

50

; 30-a

IL lo

FIG. 5. Numbers of IFN-y (l)-, IL-4 (U)-, or IL-10 (El)-producingCD4+ splenocytes from CD2F1 mice at 7 days after i.v. infection ofvirulent CA-6 cells. Mice were either untreated (naive) or were

infected with PCA-2 cells (14 days before) or were pretreated with thefractions IF or MP-F2 at 10 jig i.p. on days 16, 13, 10, and 4 beforeinfection. The numbers of cytokine-producing CD4+ cells from naiveuninfected controls were 10 + 4 for IFN-y, 15 ± 3 for IL-4, and 10 +

5 for IL-10/104 cells.

mice 7 days after challenge with CA-6, at the time at which Thpolarization has already occurred (40). As shown in Fig. 5,pretreatment with MP-F2 resulted in the presence of a highnumber of cells producing IFN--y, which was even higher thanthat found in PCA-2-vaccinated mice, so that the ratio betweenthe number of cells producing IFN--y and that of cells produc-ing IL-4 was, as in the PCA-2-vaccinated mice, inverted withrespect to the ratio calculated for naive, CA-6-infected ani-mals. However, the number of IL-4-producing CD4+ cells wasalso significantly higher in MP-F2-treated than in PCA-2-vaccinated mice (52 per 104 cells versus 24 per 104 cells,respectively; P < 0.05). The increase in the frequency ofIFN-y-producing CD4+ cells in MP-F2-immunized mice wasparalleled by an elevation in the production of IFN-y by thesplenocytes of MP-F2-immunized mice (data not shown). Nodifferences were detected in the numbers of IL-10-producingcells, which were low in both cases. Pretreatment with the IF

resulted in the presence of low numbers of IFN-y-producingcells and high numbers of both IL-4- and IL-10-producingCD4+ cells, thus reproducing the pattern seen in naive,nonpretreated CA-6-infected mice. Thus, the pattern of cyto-kine production by the animals immunized with MP-F2 andwith a low degree of protection was qualitatively similar to thatof animals vaccinated with PCA-2 cells and with a high degreeof protection, but with the important quantitative exception ofIL-4 production.

DISCUSSION

The MP-F2 fraction of C. albicans was previously character-ized for its chemical and molecular composition (45, 46). It wasfound to contain a major, antigenic target of human antican-didal cell-mediated immune (CMI) response (45). In thepresent paper, we show that MP-F2 is also a suitable antigen toelicit a potent CMI reactivity in mice with candidiasis, which isaccompanied by a low but appreciable degree of protectionagainst a lethal C. albicans infection.The ability of C. albicans to elicit or interfere with host

immune responses is believed to be mostly mediated by distinctcell wall components or some intracellular protein antigen (11,18, 20). Among surface constituents, likely candidates includemannan or MP, glucan, combinations of the two, and/or cellwall proteins or glycoproteins. It has been suggested that theimmunomodulatory function of MPs may reside in their lectin(13) or endotoxin-like (47) activities and in the molecularmimicry of host adhesion molecules (10). Since MPs arecomplex, multifunctional molecules bearing distinct saccharideand protein moieties, they are likely to induce or modulatedifferent aspects of the immune response, as exemplified by therespective roles of the two moieties as inducers of humanlymphomonocyte proliferation and neutrophil activation withcytokine production in vitro (29, 46).

In particular, an acidic MP fraction from an MP extract of C.albicans, namely, the MP-F2 fraction, was found to contain oneor more constituents which are recognized as T-cell recallantigens by human PBMC (45, 46). One active constituent wasa 65-kDa MP, which contains a large proportion of the totalprotein moiety of MP-F2. The pattern of cytokines producedby MP-F2-stimulated human PBMC was clearly compatiblewith a preferential activation of CD4+ cells of the Thl subset(5, 45). These ex vivo functions are considered to be thecorrelates of the antifungal CMI response in vivo (27), suchthat they could not be elicited in individuals with impaired

TABLE 1. Effect of MP-F2 administration on the course of subsequent C. albicans infection

In vivo pretreatment valuea Infection with CA-6 DTHAntigen

Dose Route Day(s) MSTF DST CFU (log1o)e

Vehicle 8 8/8 7.2 + 0.3 2.0 0.4PCA-2 106 cells i.v. 14 >60f 1/8f 4.5 + 0.4f 9.7 ± 1.2fMP-F2 10 ,ug i.p. 16, 13, 10, 4 169 6/8 5.6 + 0.3f 7.5 ± 2.fMP-F2 30 ,ug i.p. 16, 4 8 8/8 7.0 ± 0.8 NDhMP-F2 10 jig s.c. 16, 13, 10, 4 10 8/8 6.7 ± 0.9 NDIF 10 jig i.p. 16, 13, 10, 4 6 8/8 6.8 ± 0.2 1.8 ± 0.3

a Pretreatments were done a number of days before infection with 2 x 10' CA-6 cells i.v. On day 4, the doses of fractions injected were threefold higher.b Development of DTH reactivity to whole Candida cells was assessed 7 days after infection.c MST, median survival time (days).d D/T, number of dead animals over total number of animals injected.e CFU were determined from kidneys on day 7 postinfection.f P < 0.05, compared with mice treated with vehicle only.g P < 0.01, compared with mice treated with vehicle only.h ND, not determined.

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CMI response, such as patients with cancer (2) or HIV-infected patients (33). Because the development of antican-didal cellular immunity in mice is strictly dependent on theactivation of CD4+ Thl cells (15, 32, 35, 37, 39, 41, 42), theMP-F2 fraction was an obvious, sensible candidate for evalu-ating the ability of selected C. albicans antigens to elicitThl-mediated functions and protection in the murine model ofinfection. We were also prompted to this investigation by therecognized lack of knowledge on the immunoprotective activ-ity of specific, well-established C. albicans immunogens. Over-all, the data reported in this paper, by showing that MP-F2 (i)induces strong DTH in mice uninfected with C. albicans miceand detects DTH in C. albicans-vaccinated mice, (ii) inducessplenocyte proliferation and cytokine production in mice withcandidiasis, and (iii) induces a low but consistent degree ofprotection against a challenge with virulent C. albicans, sub-stantiate the idea that this MP fraction contains one or moremajor immunogenic constituents of C. albicans which mightplay a role in the mechanisms of anti-Candida defense.As observed with human PBMC (5, 46), MP-F2 induced the

production of IFN--y in culture supernatants of MP-F2-stimu-lated splenocytes to levels which were comparable to or even

higher than those induced with whole C. albicans cells. Theabsence or undetectable production of both IL-4 and IL-10 inthe cultures suggests that, similarly to what was observed withhuman PBMC (5), MP-F2 has the property of interacting withcells of the Thl subset. Indeed, MP-F2 induced substantialIFN-y production in culture supernatants of purified CD4'cells from vaccinated mice, which clearly suggests that theability of MP-F2 to induce IFN--y production in C. albicans-immune splenocytes is largely due to activation of Thl cells.Moreover, IFN-y production could be increased by neutraliza-tion of IL-4, but particularly IL-10, both in CD4+ cell culturesand in unfractionated splenocytes, suggesting that IL-10, whichwas not detected in culture supernatants of splenocytes stim-ulated with MP-F2, was nonetheless produced after MP-F2stimulation, even though to an undetectable level. It hasalready been reported that IL-10 adversely affects IFN--yrelease in vitro (22).The ability of MP-F2 to stimulate IFN--y-producing CD4+

lymphocytes is in line with the DTH-inducing capacity of thisfraction. Skin test reactivity to C. albicans cells and otherantigens is used to assess the levels of CMI response in vivo,such that patients suffering from severe candidiasis havedepressed CMI response and low or no DTH reactivity (28).DTH is an in vivo immune response mediated by CD4+antigen-major histocompatibility complex class II-restrictedThl cells (16), which require the presentation of an antigen inboth the sensitization and the effector phases (44). Our datashow that MP-F2 may contain antigenic epitopes which are

recognized by DTH-mediating CD4+ lymphocytes in both theafferent and the efferent phases of this response. In particular,the finding that immunization of mice with MP-F2 resulted inIFN--y production by splenocytes in vitro, an increase in thefrequency of IFN-y-producing CD4+ cells, and induction ofDTH reactivity which was transferable to naive mice, clearlyindicates that MP-F2 has the ability to induce in vivo theactivation of IFN-y-producing CD4+ Thl cells.

Since no direct inference can be made concerning anti-C.albicans protection from the ability to stimulate (or depress)DTH reactivity (24), we also examined whether immunizationwith MP-F2 induced in mice a degree of protection against a

systemic challenge by a virulent C. albicans strain. We were

encouraged in this effort by our previous report that genetically(37) or C. albicans whole-cell vaccine-induced (35, 38) resis-tance to systemic candidiasis is largely dependent on the

preferential expansion of the CD4+ Thl subset, which specif-ically produces IFN--y, as revealed by in vivo (35) and in vitro(37, 39, 41) studies. We found that vaccination of mice withMP-F2 induced a level of anti-C. albicans protection that wasclearly inferior to that achievable in the same experimentalmodel by whole-cell vaccine. Interestingly, MP-F2-inducedprotection was associated with IFN-,y production by thesplenocytes of immunized animals and a number of IFN--yproducing CD4+ cells which was even higher than that foundin mice vaccinated with PCA-2. Importantly, however, thenumber of CD4+ cells producing IL-4 was higher in mice thathad been vaccinated with the MP-F2 fraction than in those thathad been vaccinated with PCA-2. We have recently reported(40) that the levels of IFN-y do not correlate per se with theinduction of the anticandidal Thl response. As has been foundin other experimental models of infection (26), the levels ofconcomitantly expressed cytokines, such as IL-4 and IL-10,play a dominant role in the selection of the anticandidal Thphenotype in vivo. In particular, IL-4 may have a detrimentaleffect on induction of anti-C. albicans protection (32, 39).Thus, despite the elevated IFN-y production, the presence ofa substantial number of IL-4-producing CD4+ cells aftervaccination with MP-F2 may hinder the generation of anefficient, long-term-protective Thl response of the kind seenafter vaccination with low-virulence PCA-2 cells.The results described above suggest that antigenic peptides

from MP-F2 could be generated in vivo, such that bothIFN--y-producing and IL-4-producing CD4+ cells could beactivated. Because the mutually exclusive production of IL-4and IFN--y by CD4+ cells is one major discriminating factorbetween Thl and Th2 responses in murine candidiasis, theseresults also suggest that an altered balance between the twosubsets occurred in mice after MP-F2 treatment. Separation ofdistinct immunogenic constituents of MP-F2 could help todiscriminate among the MP-F2 molecules activating either oneof the two CD4+ Th subsets. In addition, the weakly protectivepotential of MP-F2 could be increased by concomitant admin-istration of cytokine antagonists. In this regard, studies areunder way to evaluate the efficacy of MP-F2 in combinedtreatments with IL-4 and IL-10 antagonists.An alternative explanation for the lack of a commensurate

relationship between DTH induction and protection aftervaccination with MP-F2 is that the DTH-mediating CD4+ cellsgenerated in response to MP-F2 do not belong to the CD4+Thl subset which mediates acquired resistance. There isalready evidence of a dissociation between DTH suppressionand preserved protection in C. albicans-immunized animalsthat have been given mannan preparations of C. albicans (20).The vigorous DTH induction by MP-F2 coupled with a weakdegree of induced protection is certainly in line with theevidence given above and clearly suggests the lack of a directcorrelation between the ability to mount a strong Candida-specific DTH response and anti-Candida protection in themurine model of infection adopted. Notably, our MP-F2preparation, which also contained >90% mannan, inducedrather than depressed (under the concentrations and theroutes of administration used) DTH reaction to Candida, andwe have preliminary evidence that DTH induction in our

model is protein (6% of the MP-F2 composition) dependent(42a). In general, a direct comparison between our results andthose obtained by previous investigators using mannan or MPpreparations is impossible because of the remarkable differ-ence in the murine model adopted and the kind of antigenicpreparations used. The diversified, sometimes opposite effectsof different MP constituents on the immune system have longbeen recognized (19, 20). Evidence of the dissociation of T

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cells mediating DTH from those mediating acquired immunityhas also been found in mice with Listeria monocytogenesinfections (48). In addition, it has been recently described thatthe Thl population may be further subdivided into at least twopopulations (31).Whatever the mechanism by which MP-F2 affects antican-

didal Th cell development in vivo, it is legitimate to assumethat it may result from the presence of different antigenicdeterminants produced by processing of MP-F2. It is alsoconceivable that the type of antigen-presenting cells firstencountered by the antigen may dictate the type of Thresponse subsequently induced, as already reported (9, 23, 24).This statement would predict that the route of the antigeninjection influences the type of Th immunity elicited. Interest-ingly, recent data (hla) show that human lymphocyte prolifer-ation by MP-F2 requires antigen processing, thus stronglysuggesting that an active antigen processing may occur in vivo.It is worth mentioning that the partially protective efficacy ofMP-F2 given in vivo is strictly dependent on the route ofadministration, with absolute preference for the peritonealone. In this regard, it has been repeatedly demonstrated thatthe i.p. route is an optimal way of inducing immunomodulatoryeffects in mice by C. albicans cell wall components (43). Finally,through its polysaccharide moiety, the MP-F2 fraction is astrong enhancer of neutrophil functions, including anti-C.albicans activity as well as proinflammatory cytokine produc-tion in vitro (14, 29). Whether this also happens in vivo and,specifically, under the conditions of our experiments, is notknown; however, it has to be considered as a potential factorinfluencing CMI response and anti-Candida protection.

In conclusion, MP-F2, a defined and relatively well-charac-terized MP fraction of the cell wall of C. albicans, is endowedwith the ability to induce CD4+ Thl cell development in mice,thus confirming its function as a recall antigen in the assess-ment of adaptive antifungal immunity in humans. Moreover, itappears that MP-F2 may represent a useful source of mole-cules to use as a tool to unravel the mechanisms underlying thegeneration or modulation of T-cell-dependent immunity andprotection against C. albicans infections.

ACKNOWLEDGMENTSThis work was supported in part by grants to F.B. and A.C. from The

National AIDS Project (Ministero della Sanita-Istituto Superiore diSanita) (contracts no. 920/U and no. 9205.02).We are grateful to Anna M. Marella and Eileen Zanetti for their

help in the preparation of the manuscript.

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