Microbes and Infection 9 (2007) 1078e1088www.elsevier.com/locate/micinf

Original article

The relative importance of CD4þ and CD8þT cellsin immunity to pulmonary paracoccidioidomycosis

Andressa P. Chiarella, Celina Arruda, Adriana Pina, Tania A. Costa,Rita C.V. Ferreira, Vera L.G. Calich*

Departamento de Imunologia do Instituto de Ciencias Biomedicas da Universidade de S~ao Paulo,Av. Prof. Lineu Prestes 1730, CEP 05508-900, S~ao Paulo, Brasil

Received 7 November 2006; accepted 26 April 2007

Available online 17 May 2007

Abstract

Protective immunity in paracoccidioidomycosis (PCM) is believed to be mediated by cellular immunity, but the role of T cell subsets hasnever been investigated. The aim of this study was to characterize the function of CD4þ and CD8þ T cells in the immunity developed by sus-ceptible, intermediate and resistant mice after P. brasiliensis infection. In susceptible mice, depletion of CD4þ T cells did not alter disease se-verity and anergy of cellular immunity but diminished antibody production. Anti-CD8 treatment led to increased fungal loads, but restored DTHreactivity. In resistant mice, both CD4þ and CD8þ T cells control fungal burdens and cytokines although only the former regulate DTH reactionsand antibody production. In the intermediate strain, deficiency of whole T and CD8þ T cells but not of CD4þ T or B cells led to increasedmortality rates. Thus, in pulmonary PCM: (a) irrespective of the host susceptibility pattern, fungal loads are mainly controlled by CD8þ T cells,whereas antibody production and DTH reactions are regulated by CD4þ T cells; (c) CD4þ T cells play a protective role in the resistant andintermediate mouse strains, whereas in susceptible mice they are deleted or anergic; (d) genetic resistance to PCM is associated with concom-itant CD4þ and CD8þ T cell immunity secreting type 1 and type 2 cytokines.� 2007 Elsevier Masson SAS. All rights reserved.

Keywords: Paracoccidioidomycosis; Fungal infection; Lung; T cells; Cytokines; T-cell deficient mice; Scid mice

1. Introduction

Paracoccidioidomycosis, the most prevalent deep mycosisin Latin America, is caused by Paracoccidioides brasiliensisa dimorphic fungus that produces conidia in its mycelialform [1,2]. Individuals from endemic areas who have inhaledmycelial fragments or fungal spores usually develop PCMinfection which is characterized by positive delayed type hy-persensitivity (DTH) reactions, production of IFN-g and IL-2by antigen-stimulated lymphocytes but absence of specific an-tibodies. Patients with the acute or severe forms of diseasepresent deficient cell-mediated immunity, high levels of anti-bodies, preferential secretion of type 2 cytokines, and loose

* Corresponding author. Tel.: þ55 11 3091 7397; fax: þ55 11 3091 7224.

E-mail address: vlcalich@icb.usp.br (V.L.G. Calich).

1286-4579/$ - see front matter � 2007 Elsevier Masson SAS. All rights reserve

doi:10.1016/j.micinf.2007.04.016

granulomatous lesions. The benign forms show preserved cellmediated immunity, prevalent type 1 immunity and low levelsof antibodies concomitant with compact and well organizedgranulomas [3e5]. In human PCM, the regulatory effect ofCTLA-4 expression on T cell proliferation and IFN-g secreti-on, as well as the presence of natural T regulatory cells werepreviously described [6,7]. P. brasiliensis antigens were alsoshown to induce lymphocyte apoptosis, a phenomenon thatcould control the number of T-reactive cells [8]. These findingsappear to indicate that complex regulatory mechanisms operatein the control of P. brasiliensis infection. Although there is nowa reasonable body of information concerning some humoral(isotypes, cytokines) aspects of the disease, the relative func-tion of T cell subsets in the human disease is less understood.Current knowledge is based on correlative evidence that comesfrom experiments aimed to identify T cells that respond to

d.

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P. brasiliensis antigens in vitro and the relative number of Tcell subsets in peripheral blood [4e8].

Our studies on genetic susceptibility to P. brasiliensis in-fection characterized B10.A, C57Bl/6 and A/J as susceptible,intermediate and resistant strains of mice, respectively [9e11]. In the i.p. and i.t. models of infection the progressiveand disseminated disease of B10.A mice was associated withDTH anergy, absence of macrophage activation, and high fun-gal loads in non-organized lesions mimicking the severe formsof the human disease. On the contrary, A/J mice presented a re-gressive disease with well organized lesions containing lownumber of yeasts, positive cellular immunity and macrophageactivation [9e11]. Further studies on cytokine production byantigen-stimulated lymph node cells from i.p. infected miceallowed us to verify that resistance was associated with prev-alent production of IL-2 and IFN-g and susceptibility with ab-sence of type 1 cytokines. However, a typical Th2 pattern wasnot characterized since IL-4 was never detected [12]. Studieson pulmonary cytokines after i.t. infection showed that suscep-tible mice secreted higher levels of IFN-g, IL-5, and IL-10than resistant animals, indicating a mixed pattern of immuno-logical activation [11e13].

Previous in vivo studies using cytokine-depleted and genet-ically deficient mice with targeted deletion of cytokines genes(knockout, KO mice) showed that protective immunity in PCMwas associated with type 1 cytokines whereas IL-10 and IL-4deficiencies resulted in mild pathology [13e18]. In contrast,in susceptible B10.A mice IL-4 depletion led to a more severedisease [16] and early administration of recombinant IL-12caused enhanced lung pathology due to the intense inflamma-tory reaction at the infection site [18]. Furthermore, nitric ox-ide, the most important macrophage activating mediator forP. brasiliensis killing [19] was shown to have a dual role, isprotective but its excessive secretion induces depressed cellularimmunity [20e21]. As a whole, these studies showed that theTh1/Th2 hypothesis was only partially correct [11,15] andmore complex mechanisms appear to regulate genetic suscep-tibility to PCM.

As in human disease, there is scarce information on the roleof T cell subsets in murine PCM. The only study on T cell de-pletion showed that CD8þ T cells control disease severity inboth susceptible and resistant mice to P. brasiliensis infection[22]. Furthermore, to our knowledge, no studies addressing therole of CD4þ T cells have been performed. Hence, the aim ofthis investigation was to determine the relative contribution ofCD4þ and CD8þ T cells in the control of pulmonary PCM.

2. Materials and methods

2.1. Mice

Unless otherwise stated, groups of 7e10 male mice (8e1 weeks old) from the susceptible (B10.A) and resistant (A/J)strains were used to each period of infection. Male CD4-deficient (B6.129S2-Cd4tm1MAK), CD8a-deficient (B6.129S2-Cd8atm1MAK), T-cell deficient (C57BL/6nu/nu), with combineddeficiency of T and B cells (SCID-B6CB17-Prkdcscid/sjz) and

WT C57CBL/6 mice, originally obtained from Jackson La-boratories, were also used. All the animals were bred underspecific pathogen-free conditions in our animal facilities. Pro-cedures involving animals and their care were conducted inconformity with national and international policies.

2.2. Paracoccidioides brasiliensis

Pb18, a highly virulent isolate, was used throughout thisinvestigation. Pb18 yeast cells were maintained by weeklysub-cultivation in the semisolid culture medium as describedelsewhere [10]. The yeast cells were washed in phosphate-buffered saline (PBS; pH 7.2) and adjusted to 20 � 106 cells/ml based on hemacytometer counts. Viability was determinedwith the Janus green B vital dye (Merck, Darmstadt, Germany)and was always higher than 80%.

2.3. Intratracheal inoculation of P. brasiliensis

Mice were anesthetized and submitted to i.t. P. brasiliensisinfection as previously described [10]. Briefly, after i.p. anes-thesia the animals were infected with 1 � 106 P. brasiliensisPb18 yeast cells, contained in 50 ml of PBS, by surgical i.t. in-oculation that allowed dispensing the fungal cells directly intothe lungs. The skin of the animal was then sutured and themice were allowed to recover under a heat lamp.

2.4. In vivo depletion of CD4þ andCD8aþ T cells by MAbs

GK1.5 (rat IgG2b anti-CD4) and H-35 (rat IgG1 anti-CD8)hybridomas were kindly provided by Dr Ises A. Abrahamsohn(University of S~ao Paulo, Brazil). The hybridomas were grownin BALB/c nu/nu mice. Monoclonal antibodies (MAbs) werepurified from ascites as previously described [22] and assayedfor purity by sodium dodecyl sulfate-polyacrylamide gel elec-trophoresis. Groups of B10.A and A/J mice were given 200 mgof MAbs or normal rat IgG (controls) by the i.p. route, 48 and24 h before infection and 100 mg of the MAbs or rat IgGweekly thereafter, for 8 weeks. Another protocol was used todeplete CD4þ T cells: 1.0 mg of GK1.5 MAb or normal ratIgG was injected by the i.p. route 48 and 24 h before infectionand 500 mg of the MAb or rat IgG weekly, for 8 weeks. Miceonly infected as well as non-infected and non-depleted ani-mals were also used as controls.

2.5. Spleen cells preparation and cytofluorometry

Before infecting mice, the efficiency of the in vivo MAbtreatment on the profile of lymphocyte subpopulations was de-termined by flow cytofluorimetric analysis. Groups of B10.Aand A/J mice received two injections of anti-CD4, anti-CD8MAb or normal rat IgG and 48 h after treatment (day 0 of in-fection) their spleen were removed and homogenized to obtainsingle cell suspensions. Cell suspensions of each animal werewashed, adjusted to 5 � 106 viable cells/ml and stained withphycoerytrin-conjugated anti-CD4, anti-CD8 or rat anti-mouse

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Ig MAb (Pharmingen, San Diego, CA). The stained cells wereanalyzed immediately on a FACScan equipment using thePC-Lysys software (Becton&Dickinson, CA) gating on lym-phocytes as judged from forward and side light scatter. Tenthousand lymphocytes were counted and the data were ex-pressed as the number of viable CD8þ T, CD4þ T and Igþ

cells. The efficiency of the CD8þ and CD4þ T cell depletionwas assessed by calculating the decrease of this subpopulationrelative to the normal IgG-treated animals.

2.6. Survival and organ colony formingunits (CFU) assays

The number of viable microorganisms in the lungs, liverand spleen from infected mice was determined by countingthe number of CFU. Eight to ten animals from each mousestrain and treatment were sacrificed at several post-infectionperiods and the enumeration of viable organism was done bycounting the number of CFU, as described elsewhere [10].For survival assays 8e10 mice were infected as describedand monitored for survival over 250 days. CFU and mortalityexperiments were repeated two or three times.

2.7. Specific antibody levels

Levels of P. brasiliensis specific isotypes (IgG1, IgG2a andIgG2b) were measured by a previously described ELISA em-ploying a cell-free antigen [10] prepared from a pool of differ-ent P. brasiliensis isolates (Pb 339, Pb 265, and Pb 18).

2.8. Cytokine analysis

The levels of IFN-g, IL-2, IL-4, IL-5 and IL-10 were mea-sured in lung supernatants by capture ELISA using antibodypairs purchased from Pharmingen (San Diego, CA). TheELISA procedure was performed according to the manufactur-er’s protocol.

2.9. Delayed-type hypersensitivity (DTH assay)

The DTH reactions were evaluated just before sacrifice of thesame animals used in the CFU assays, by the footpad test ac-cording to previously determined conditions [23]. Briefly,mice were inoculated with 25 ml of the Fava Nettos’s antigen[23] and the footpad thickness was measured immediately be-fore and 24 h after antigen inoculation. Non-infected mice sub-mitted to the footpad test were used as controls.

2.10. Statistical analysis

All values are means � SEM, unless otherwise indicated.Differences between two means were evaluated by the Stu-dent‘s t-test. Differences between three means were evaluatedusing a one-way ANOVA, followed by multiple comparisonsaccording to Tukey’s procedure. Differences between survivaltimes were determined with the LogRank test using GraphPad

Prism v3 for Windows (GraphPad Software). A p value <0.05was considered significant.

3. Results

3.1. Effect of in vivo MAb treatment on splenic T cells

Groups of B10.A and A/J mice were injected i.p. with anti-CD8, anti-CD4 MAb or control IgG at days �2 and �1. Atday 0 they were sacrificed, their spleens removed and lympho-cytes analyzed by flow cytometry. In both mouse strains, MAbtreatment resulted in 99e97% depletion of CD4þ or CD8þ Tcells, and in greatly altered CD4/CD8 ratios (Fig. 1). Anti-CD4 treatment decreased CD4/CD8 ratios to 0.02 for resistantand susceptible mice, whereas profoundly augmented ratioswere detected after anti-CD8a MAb treatment (54.9 and35.7 for A/J and B10.A mice, respectively). We have also an-alyzed spleen cells at week 8 of infection of MAb-treated andcontrol (IgG treated) mice. Treatment with GK1.5 led to 96%reduction of CD4þ T cells from both mice strains whereas H-35 MAb reduced 43% and 58% of CD8þ T lymphocytes fromA/J and B10.A mice, respectively (data not shown).

3.2. Anti-CD4 treatment alters fungal loads and DTHreactions of resistant but not of susceptible mice

At day zero of MAb or IgG treatment, B10.A and A/Jmice were infected and further analyzed by CFU assays.At week 4 of infection CD4-depleted resistant mice pre-sented increased fungal loads in the liver indicating an earlyprotective effect of these cells. At week 8, decreased fungalloads were recovered from lungs of anti-CD4-treated A/Jmice revealing that CD4þ T cells exerted a deleterious effectin the control of fungal growth. Surprisingly, anti-CD4 treat-ment did not modify the organ CFU counts of susceptiblemice (Fig. 2A).

Depletion of CD4þ T cells led to decreased DTH reactionsof resistant mice, but did not modify the low reactivity oranergy of B10.A animals (Fig. 2B). Thus, CD4þ T cells ap-pear to regulate the disease of resistant but not of susceptiblemice.

We have also performed experiments to verify if a moredrastic anti-CD4 treatment would alter immunity of suscepti-ble mice. Thus, in vivo, B10.A and A/J mice were depletedwith 1.0 mg of GK1.5 MAb at days �2 and �1 of infectionand 500 mg per week thereafter. Confirming the previousschedule with small doses of GK1.5, at week 8 resistantmice presented decreased number of viable yeasts in the lungsand negative DTH reactions. Even with this high doses proto-col susceptible mice did not modify their fungal burdens andDTH anergy (data not shown).

3.3. CD8aþ cells control fungal loads of resistant andsusceptible strains and DTH anergy of susceptible mice

CD8-depleted mice were analyzed only at week 8 of infec-tion since in our previous work the effect of CD8þ T cell

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depletion was studied at earlier post-infection periods [22].Confirming our previous results, depletion of CD8þ T cellsled to a more severe disease in both susceptible and resistantmice. Increased fungal loads were seen in the lungs ofB10.A and A/J mice and in the liver of susceptible animals.These data showed that in both mouse strains CD8þ T cells

Fig. 1. Anti-CD4 and anti-CD8 MAb treatments of susceptible (B10.A) and

resistant (A/J) mice to P. brasiliensis cause a marked depletion of T cells

and alter CD4/CD8 ratios. Groups of B10.A and A/J mice (n ¼ 4) were given

200 mg of MAbs or normal rat IgG (controls) by the i.p. route, 48 and 24 h be-

fore sacrifice. Their splenic cells were collected and analyzed by flow cytom-

etry to determine lymphocyte subpopulations. As shown, both MAb

preparations were efficient and significantly altered the CD4/CD8 T cells ra-

tios. Upper figures in the first two graphs denote percentage of depletion

and in the third one the CD4/CD8 ratios after T cell depletion.

play an important role in the control of fungal burdens(Fig. 3A).

When DTH response was studied after CD8aþ T cell deple-tion, no effect on the cutaneous reactivity of resistant mice wasobserved but, interestingly, a reversal of DTH anergy was de-tected in B10.A mice (Fig. 3B).

3.4. CD4þ but not CD8þ T cell depletion induces markeddecreases in antibodies production.

Antibodies appear not to play a protective role in humanand murine PCM although Th1/Th2-regulated isotypes havebeen used as good markers of the ongoing immune responses[3,4,10,12]. In A/J mice, CD4 depletion led to decreased levelsof IgG2a, IgG1 and IgG2b antibodies indicating that IFN-g,IL-4 and TGF-b-regulated isotypes are controlled by CD4þ

T cells. Equivalent results were obtained with B10.A micebut no differences in the levels of IgG2a were detected(Fig. 4). In the present work we could also confirm thatCD8þ T lymphocytes do not control antibody production sincein both mouse strains no differences in isotype levels werefound (data not shown).

3.5. Effect of CD4þ cell depletion on pulmonarytype 1 and type 2 cytokines

The levels of type 1 and type 2 cytokines were measured inlung homogenates. Decreased production of IL-2 was verifiedin CD4-depleted A/J mice at week 8 of infection but no differ-ences were observed with the susceptible strain (Fig. 5A). Re-garding type 2 cytokines, a significant decrease of IL-4, IL-5and IL-10 was detected at week 8 post-infection in lung ho-mogenates of CD4-depleted A/J mice indicating that thesecells exerted in untreated mice a clear type 1 immune re-sponse. On the contrary, CD4þ T cells did not regulate pulmo-nary type 2 cytokines of susceptible mice (Fig. 5B).

3.6. Effect of CD8aþ cell depletion on pulmonarytype 1 and type 2 cytokines

To extend our previous studies on the role of CD8þ T cellsin murine PCM, cytokines were measured at week 8 afterMAb treatment. As can be seen in Fig. 6, CD8þ T cellshave an opposite effect in susceptible and resistant strains.While anti-CD8 treatment led to decreased levels of IL-2,a type 1 cytokine, in the lungs of A/J mice (Fig. 6A), inB10.A animals increased levels of IFN-g indicate the presenceof a type 2 CD8þ T cell subset. Compared with untreatedmice, higher levels of IL-4, IL-5 and IL-10 were seen inCD8-depleted A/J mice demonstrating that a type 1 CD8þ Tcell was negatively regulating these cytokines. In susceptiblemice no differences in type 2 cytokines were found(Fig. 6B). As a whole, our data suggest that CD8þ T cellsof resistant mice probably have a type 1 phenotype whereasa not typical profile was found in susceptible mice.

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Fig. 2. Effect of CD4þ T cell depletion in the fungal loads and DTH reactions of susceptible (B10.A) and resistant (A/J) mice infected with P. brasiliensis. A/J and

B10.A mice were in vivo depletion of CD4þ T cells by anti-CD4 MAb (20 mg of MAbs GK1.5 by the i.p. route, 48 and 24 h before infection and 100 mg of the

MAbs weekly thereafter) and infected i.t. with one million P. brasiliensis yeasts. (A) Recovery of CFU from the lungs, liver and spleen of infected mice treated

with normal rat IgG (control) or anti-CD4 MAb were shown. The results are representative of three independent experiments. The bars depict means � SE of log10

CFU obtained from groups of 8e10 mice at weeks 4 and 8 after infection. (B) DTH reactions were evaluated at weeks 4 and 8 in CD4-depleted and control mice.

Mice were injected intrafootpad with a soluble yeast antigen 24 h before measurement of the footpad response. The bars depict means � SE of footpad swelling.

The dotted line denotes means � 2SD (confidence interval, 95%) of non-infected B10.A and A/J mice submitted to the footpad test (n ¼ 9 mice). #p < 0.001;

*p < 0.05, significantly different from IgG-treated groups.

3.7. Combined deficiency of T and B cells (SCID mice),whole T cells (nude mice) and CD8þ T cells(CD8�/� mice) result in more severePCM in C57Bl/6 mice

Due to the extremely chronic nature of PCM, the impairedneutralization of CD4þ and CD8þ T cells caused by theincreased resistance of activated T cells and the immuneresponse of hosts to a sustained MAb treatment, genetically

immunodeficient mice were also used to characterize the influ-ence of T cell subsets in PCM. In addition, these experimentscould expand the knowledge on the role of T cells in PCM be-cause the C57BL/6 strain was shown to have an intermediatepattern of susceptibility to P. brasiliensis infection [9]. Thus,C57Bl/6 WT mice and their deficient counterparts devoid ofT and B lymphocytes (SCID mice), T cells (nu/nu mice)CD4þ and CD8aþ T cells (CD4�/� and CD8�/�) were i.t. in-fected and disease severity analyzed by mortality and CFU

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Fig. 3. Effect of CD8þ T cell depletion in the fungal loads and DTH reactions of susceptible (B10.A) and resistant (A/J) mice infected with P. brasiliensis. A/J and

B10.A mice were in vivo depleted of CD8þ T cells by anti-CD8 MAb (200 mg of MAbs H-35 by the i.p. route, 48 and 24 h before infection and 100 mg of the

MAbs weekly thereafter) and infected i.t. with one million P. brasiliensis yeasts. (A) Recovery of CFU from the lungs, liver and spleen of infected mice treated

with normal rat IgG (control) or anti-CD8 MAb were shown. The results are representative of two independent experiments. The bars depict means � SE of log10

CFU obtained from groups of 8e10 mice at week 8 after infection. (B) DTH reactions were evaluated at week 8 in CD8-depleted and control mice. Mice were

injected intrafootpad with a soluble yeast antigen 24 h before measurement of the footpad response. The bars depict means � SE of footpad swelling. The dotted

line denotes means � 2SD (confidence interval, 95%) of non-infected B10.A and A/J mice submitted to the footpad test (n ¼ 9 mice). #p < 0.01; *p < 0.05, sig-

nificantly different from IgG-treated groups.

assays. It can be seen in Fig. 7 that SCID and nude mice werethe most susceptible strains to P. brasiliensis infection, fol-lowed by CD8-deficient animals. The mean survival times(MST) of SCID and nude mice were very similar and signifi-cantly different from all other studied strains. Compared withWT controls, CD8�/� but not CD4�/� mice present signifi-cantly diminished survival times. In addition, CD4-deficientmice were shown to be more resistant than the CD8-deficientcounterparts. The MST of SCID, nude, CD8�/�, CD4�/� andWT mice were 59, 73, 193, 227 and higher than 250 days,respectively.

To further confirm mortality studies, CFU data were ob-tained from infected immunodeficient mice. Compared withWT controls, SCID mice presented increased CFU counts inall organs and periods assayed (Fig. 7). At week 8, an

impressive number of viable yeasts was detected in the liversof SCID mice (5.35þ/�0.07 log10) when compared with WTmice (0.65þ/�0.41 log10). The difference of viable yeasts re-covered was close to 5 log10 CFU for livers and higher than2 log10 for spleens.

By week 4 of infection, and compared with their WT con-trols, no differences in organ fungal loads were detected inCD4- and CD8-deficient mice (data not shown). However, atweek 8 higher number of yeast cells was seen in the liverand spleen of CD4�/� and CD8�/� mice, respectively. Byweek 12 of infection, increased number of yeasts was detectedin the lungs, liver and spleen of CD8�/� mice while no signif-icant differences were observed in CD4�/� mice. These datareinforce our findings with T cell depletion and indicate a prev-alent protective effect of CD8þ T cells.

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Fig. 4. CD4þ T cell control antibody production by resistant and susceptible mice to P. brasiliensis infection. Levels of fungal specific isotypes in the sera of CD4-

depleted (GK1.5) and control (IgG-treated) B10.A and A/J mice at weeks 4 and 8 after i.t. infection with 106 yeast cells. Sera were assayed for IgG1, IgG2a, and

IgG2b antibodies by using an isotype-specific ELISA as described in Section 2. The bars depict means (log2) and SE of serum titers for 8e10 mice per group.

#p < 0.001; þp < 0.01; *p < 0.05, significantly different from IgG-treated groups.

4. Discussion

To our knowledge, studies on the role of T cell subsets inthe immune response to P. brasiliensis infection were neveraddressed. Therefore, in the present investigation a well-defined experimental model using resistant, intermediate andsusceptible mice strains, one single fungal isolate and twoexperimental approaches were used to understand the mainregulatory and effector functions of T cell subpopulations inPCM. The use of T cell-depleted and T cell-deficient mice al-lowed us to obtain complementary information since each oneof these approaches has their own limitations. As a whole, ourdata demonstrated that both CD4þ and CD8þ T cells are pro-tective to pulmonary PCM, but the latter have a prevalent role.The contribution of each T cell subset in the immune responsedepends on the genetic pattern of hosts and, a balanced CD4/CD8 T cell immunity, regulating type 1 and type 2 cytokinesecretion, correlates with host resistance to P. brasiliensis in-fection. Furthermore, susceptibility could not be linked to Th2immunity but with anergy or deletion of CD4þ T lymphocytes.

Our findings demonstrated that the severity of P. brasiliensisinfection in susceptible B10.A mice is not regulated by CD4þ

T cells. In CD4-depletion experiments with low (Fig. 1) andhigh doses (data not shown) of anti-CD4 MAb no alterationsin CFU counts and no typical cross regulation by type 1 ortype 2 cytokines were seen, indicating the anergy (or deletion)of CD4þ T cells. Thus, the relative protection of susceptiblemice is mainly mediated by CD8þ T cells. In this aspect, wehave several data indicating that the natural immunity of sus-ceptible mice could induce this pattern of acquired immunity.Early in infection, B10.A mice are more able than resistantmice to control fungal loads [10]; their alveolar macrophageshave efficient fungicidal ability and secrete high levels of IL-12 and nitric oxide [15]. The elevated secretion of nitric oxide

would induce CD4þ T cell apoptosis, a fact previously de-scribed by us and others in experimental PCM [20,21]. The se-cretion of high IL-12 levels associated with elevated expressionof costimulatory molecules (Pina and Calich, unpublishedresults) would preferentially prime CD8þ T cells [24] whichbehave as the main T cell subset controlling fungal growth(Calich and Blotta) [15]. In agreement, previous results ofour laboratory demonstrated that P. brasiliensis-activated den-dritic cells (DC) of B10.A mice express costimulatory mole-cules, secrete high levels of IL-12 and NO, but induce poorlymphocyte proliferation (PinaandCalich, unpublished results).This behavior would explicate the CD4þ T cell anergy (in-duced by NO or activation-induced apoptosis) and the protec-tive immunity mainly mediated by CD8þ T cells. However,the increased fungal burdens associated with positive DTH re-actions induced by anti-CD8 treatment are difficult to explain.Nevertheless, the current knowledge on the biology of CD8aþ

cells opens an interesting perspective to understand our results.By limiting available tryptophan, and inducing tryptophan me-tabolites, indoleamine oxygenase (IDO)-expressing CD8aþ

dendritic cells are able to control the multiplication of trypto-phan-dependent microorganisms and to induce apoptosis of an-tigen-specific CD4þ T cells, respectively [25e27]. It can besupposed that tolerogenic CD8aþ DC expressing IDO havemediated the T cell anergy (or deletion) and the control of fun-gal growth here observed. Besides depleting CD8þ T cells, theanti-CD8a MAb would have also depleted this regulatory DC.Although our work does not bring data to support this hypoth-esis, the putative presence of a tolerogenic CD8aþ DC couldconciliate the increased fungal loads, the augmented IFN-g se-cretion and the reversal of DTH anergy observed here.

Depletion experiments in resistant mice demonstrated thatCD4þ T cells do control severity of the disease as well as cy-tokine and antibody secretion. Although we have no direct

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Fig. 5. Effect of CD4þ T cell depletion on the levels of type 1 (A) and type 2 (B) cytokines in lung homogenates of susceptible (B10.A) mice and resistant mice

(A/J). At weeks 4 and 8 after i.t. infection with 106 yeast cells of P. brasiliensis, lungs from CD4-depleted (GK1.5) and control (IgG-treated) B10.A and A/J mice

were collected and disrupted in 5.0 ml of PBS and supernatants were analyzed for cytokines content by capture ELISA. The bars depict means � SE of cytokine

levels (8e9 animals per group). C denotes means � SE of cytokine level in the lung of non-infected B10.A and A/J mice (n ¼ 7 mice). #p < 0.001, compared with

control mice.

data on the phenotype of cytokine secreting cells and severallung infiltrating inflammatory cells can secrete these mediators,some inferences can be drawn from the major alterationscaused by T cell depletion. In A/J mice, at week 4 of infectiona protective CD4þ T cell which induces DTH reactions and di-minishes hepatic fungal loads was detected. At week 8, how-ever, a dual function appears to be exerted by this T cellsubset. The decreased CFU counts associated with diminishedlevels of IL-4, 5 and 10 in the lungs of anti-CD4-treated micesuggest the previous presence of a type 2 CD4þ subpopulation;the concomitant decrease in DTH reactions and the diminishedlevels of IL-2 indicates, however, the presence of a type 1CD4þ T cell subpopulation. Besides pulmonary cytokines,

studies on antibody production reinforced the possibility ofa mixed Th1/Th2 response (Fig.4). Indeed, at week 8 of infec-tion CD4-depleted A/J mice presented diminished synthesis ofTh1 and Th2 regulated isotypes (IgG2a, IgG1 and IgG2b). Al-ternatively, we could hypothesize that anti-CD4 treatment ledto a decreased number of natural regulatory T cells, which usu-ally secrete IL-10 and/or TGF-b [28], resulting in increasedfunction of protective type 1 CD8þ T cells. The latter cellswould reduce fungal loads and negatively regulate IL-4 andIL-5 synthesis. This possibility, however, does not explain theconcomitant decrease in IL-2 and DTH reactions. Both hypoth-eses, which are not mutually exclusive, comprise pros and consand have to be clarified by further studies; however, our

1086 A.P. Chiarella et al. / Microbes and Infection 9 (2007) 1078e1088

Fig. 6. Effect of CD8þ T cell depletion on the levels of type 1 (A) and type 2 (B) cytokines in lung homogenates of susceptible (B10.A) mice and resistant mice (A/

J). At week 8 after i.t. infection with 106 yeast cells of P. brasiliensis, lungs from CD8-depleted (H-35) and control (IgG-treated) B10.A and A/J mice were col-

lected and disrupted in 5.0 ml of PBS and supernatants were analyzed for cytokines content by capture ELISA. The bars depict means � SE of cytokine levels (7e

9 animals per group). C denotes means � SE of cytokine level in the lung of non-infected B10.A and A/J mice (n ¼ 7 mice). *p < 0.05 significantly different from

IgG-treated groups.

findings primarily suggested the concomitant function of cellswith type 1 and type 2 phenotypes, but do not exclude the par-ticipation of other effector or regulatory cells.

In resistant mice, CD8þ T lymphocytes were able to controlfungal loads and cytokine secretion. The decreased levels ofIL-2 and the increased amounts of pulmonary IL-4 and IL-5observed after anti-CD8 treatment suggest that this T cell sub-population has a preferential type 1 phenotype. So, genetic re-sistance to PCM appears to be dependent on the function ofCD8þ type 1 T cells in cooperation with protective CD4þ Tcells. It is possible that an early type 1 activity mediated byCD4þ T cells could govern the polarization of CD8þ T cellsto a stable type 1 phenotype [24].

We have also explored the function of T cell subsets inC57BL/6 mice which has an intermediate pattern of suscepti-bility to P. brasiliensis infection. As human PCM has a gamut

of clinical manifestations ranging from mild to severe diseases[1e5], the studies with this intermediate strain appear to bettercover the vast possibilities of T cell functions in genetically di-verse hosts. Furthermore, the use of a genetically deficientstrain of mice would avoid the partial or temporal effects ofMAb treatments.

Studying CD4- and CD8-deficient mice we could confirmthe prominent protective effects of CD8þ T cells already indi-cated by depletion experiments, and the efficient innate immu-nity of hosts infected with P. brasiliensis: differences in CFUcounts were seen only after 8 weeks of infection. Interestingly,in pulmonary PCM the lack of CD4þ or CD8þ T cells was notas dramatic as observed in other fungal pathologies [29,30]. Inaddition, as described for immunodeficient mice in other fun-gal infections [31e33], each cell subset appears to replace theeffector activities of the lacking one. The especially severe

1087A.P. Chiarella et al. / Microbes and Infection 9 (2007) 1078e1088

Fig. 7. Upper panel: survival time of T- and B-cell deficient (SCID), T cell deficient (nu/nu), CD4-deficient (CD4�/�), CD8-deficient (CD8�/�) and WT C57Bl/6

mice after i.t. infection with 1 � 106 P. brasiliensis yeast cells. The results are representative of three independent experiments. #Significantly different from all

mice strains; *significantly different from WT and CD4�/�. Lower panel: CFU counts obtained from the lungs, liver and spleen of SCID, CD4�/�, CD8�/�, and

WT C57BL/6 mice at several time points after infection with one million P. brasiliensis yeasts. #p &lt 0.001; *p < 0.05; þp < 0.01, significantly different from

WT mice.

disease of nude and SCID mice appears to reinforce this inter-pretation since the concurrent absence of CD4þ and CD8þ Tcells led to uncontrolled fungal growth and precocious mortal-ity of mice. Moreover, it could be seen that B cell deficiencydid not contribute to this disease outcome since equivalent sur-vival times were observed with SDID and nude mice. Indeed,both in murine and human PCM there are solid data demon-strating that B cell activation is associated with disease sever-ity and not with immunoprotection [1,2,15].

As a whole, our results appear to reveal the important role ofCD8þ T cells in the immunoprotection of pulmonary PCM andsuggested that this T cell subset might not be neglected in furtherstudies on cellular immunity in PCM. The function of CD4þ Tcells, however, appears to significantly vary among hosts withdifferent susceptibility patterns. Finally, this unsuspected im-portance of CD8þ T cells perhaps could explain the low fre-quency of PCM among AIDS patients [34], a finding thatdistinguishes this fungal infection from other endemic and op-portunistic mycoses whose incidence substantially increased af-ter AIDS epidemics.

Acknowledgements

This work was supported by grants from Fundac~ao de Am-paro a Pesquisa (FAPESP) e Conselho Nacional de Pesquisas(CNPq).

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