Medical Mycology February 2004, 42, 15 /25

Resistance to Cryptococcus neoformans infection in the absence of CD4 T cells

KAREN AGUIRRE*,$, JASON CROWE$, AMY HAAS$ & JANEL SMITH$ *Trudeau Institute, Saranac Lake and $Clarkson University, Potsdam, New York, USA

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Previous studies of Cryptococcus neoformans infection have revealed a role for CD4' T cells and CD8 ' T cells in anticryptococcal resistance in the lungs, but such a role has been revealed only for CD4' T cells in the brains of experimentally infected mice. In this study, we found that mice genetically engineered to lack CD4' T cells could be successfully vaccinated to express resistance to a rechallenge with Cryptococcus neoformans , provided the challenge dose was kept to lower than 1000 organisms per mouse. The challenge infection was uniformly lethal for unvaccinated control mice. Depletion of CD8 ' T cells weakened this resistance to re-challenge: both na ve and vaccinated mice that were treated with antibody raised against CD8' T cells died significantly earlier than did mice that received an irrelevant control antibody. In vitro, purified CD8' T cells taken from draining lymph nodes of antigen-experienced mice were less efficient than were identically prepared CD4' T cells at stimulating the cells of a transformed microglial cell line to inhibit C. neoformans proliferation, possibly mirroring the inferiority of CD8 ' T-cell-mediated protection observed in vivo . RNase protection assays showed similar IFN-g mRNA levels in both lymphocyte subsets. Class II major histocompatibility antigen expression was up-regulated strikingly on microglia cultured with IFN-g, but class I expression was less dramatically affected. Therefore microglial cell interaction may be more greatly enhanced with CD4 ' cells than with CD8 ' cells. Keywords CD8' T cells, Cryptococcus neoformans , microglia

Introduction
Cryptococcus neoformans is a ubiquitous fungus that causes cryptococcal meningoencephalitis in AIDS patients and other immunocompromized individuals [1,2]. In the mouse model, inhaled organisms first colonize the lung, but may metastasize to other organs, most notably, the brain and spinal cord [2]. In the lung, both CD4' and CD8' T cells are required to clear the infection. If yeasts escape and colonize the brain, rapid proliferation leads to serious central nervous system (CNS) disease [2,3]. Working with T- and B-celldeficient severe combined immunodeficient (SCID)

Received 7 January 2003; Accepted 3 July 2003 Correspondence: Karen Aguirre PhD, Department of Biology, Box 5805, Clarkson University, Potsdam, NY 13699, USA. Tel.: '/315 268 4498; Fax: '/315 268 7118; E-mail: kmaguirr@clarkson.edu

mice and wild-type counterparts, this laboratory has demonstrated a CD4' T-cell-mediated resistance to cryptococcal organisms that colonize the brain [3]. In previous studies, depletion of CD8 ' T cells had no detectable effect on T-cell-mediated resistance in vivo in brain. In contrast, CD8 ' T cells play a well-defined and important role of in containment of pulmonary infection. Immunocompetent mice can be vaccinated so that they express a potent, sterilizing immunity to an ordinarily lethal cryptococcal challenge infection [3]. The anti-cryptococcal T-cell memory response resides in the CD4' T-cell compartment. Moreover, adoptive immunization of SCID mice with CD4' T cells from immune donor mice confers anti-cryptococcal resistance on SCID recipients, as measured by reduced brain fungal burden at 10 days post challenge, and is also associated with a significantly longer survival time
DOI: 10.1080/1369-378032000141732

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in immune CD4 ' T-cell-reconstituted mice than in control mice [3]. Other researchers have also reported CD4' T-cell-mediated resistance to cryptococcal brain infection [4]. Recently, experiments in this laboratory have shown that major histocompatibility class (MHC) II expression on microglial cells of the CNS is required if resistance is to be conferred on recipient mice in similar adoptive immunization assays [5]. The class II molecule provides the proper context in which antigen is presented to specifically responding CD4' T cells [6]. It is postulated that immune surveillance of the CNS is somewhat limited by the inability of na ve T cells to cross the blood-brain barrier [7]. However, antigenprimed or activated T cells express cell adhesion molecules which facilitate their entry into the CNS. T-cell trafficking through the brain is transient, and most T cells quickly exit. Only antigen-experienced T cells which again recognize their specific antigen, in the proper MHC context, remain or proliferate within the CNS [7]. CD4 ' T cells must interact with MHC IIbearing antigen presenting cells (APC) and CD8 ' T cells must interact with MHC I bearing APC [6]. The requirement for expression of MHC II on microglia if resistance to C. neoformans brain infection is to be expressed underlines the importance of the CD4' Tcell compartment in the resistance of immunocompetent animals to meningoencephalitis. The following study was undertaken to determine whether the requirement of CD4' T cells is absolute, or whether situations exist wherein other cell types can substitute for CD4' T cells. Reasoning that CD8 ' T cells, if produced and matured in the complete absence of CD4 ' T cells, might perform some of the functions normally mediated by CD4 ' cells, we tested whether MHC class II-deficient CD4 Ab null mice could be vaccinated to resist C. neoformans brain infection. We also considered the alternative hypothesis that even a relatively modest anticryptococcal function inherent in CD8 ' T cells might be more easily detectable when CD4' cells were eliminated. The demonstration of a protective role for CD8 ' T cells might encourage research into the rational design of a vaccine engineered to elicit CD8' T-cell memory. The study might also further illuminate the mechanisms deployed by the immunocompetent animal to control cryptococcal brain infection. We demonstrate here a CD8 ' T-cell-dependent resistance to low-dose C. neoformans brain infection. Data are also presented showing that, in vitro, CD8 ' T cells may have fewer productive interactions with microglial cells of the CNS than do CD4' T cells, suggesting a possible mechanistic explanation for the

relative inefficiency of CD8 ' T cells in anticryptococcal activity.

Materials and methods

Mice
C57BL6/J inbred mice were obtained from the Animal Breeding Facility of Trudeau Institute (Saranac Lake, NY, USA), and maintained under standard husbandry conditions. Ab null mice (genetically engineered to lack expression of major histocompatibility II molecule, and therefore, also of CD4 ' T cells) [8], and back-crossed for 10 generations to the C57BL6/J mice, were the gift of Robert North and Susan Swain of Trudeau Institute. These immunodeficient mice were maintained in sterile isolator cages with high-efficiency particulate air (HEPA) filtered air supply and given sterile food and water. Wild-type mice received commercial rodent chow and acidified water. Mice were free of common pathogens, as evidenced by periodic serological testing performed by the Research Animal Diagnostic and Investigative Laboratory of the University of Missouri, Columbia, MO, USA. All protocols involving live mice were approved by the Trudeau Institutes Animal Care Committee.

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Yeast
Several strains of C. neoformans were used in this study. Cap 67 (ATCC 52817, American Type Culture Collection, Manassas, VA, USA) is an avirulent acapsular mutant. Strain 52 D (ATCC 24067) is a moderately virulent encapsulated serotype D strain. Strain 145 (ATCC 62070) is a highly virulent encapsulated serotype A. Strain 184 is a moderately virulent, lightly encapsulated serotype A strain which was the gift of JW Murphy [9] Ura5 , an avirulent heavily encapsulated uracil auxotroph of C. neoformans, was the gift of K. J. Kwon-Chung. [10]. All strains except Ura5 were maintained on Sabouraud dextrose (Sabdex) agar as previously described [3]. Ura5 was maintained on yeast enriched peptone digest medium as previously described [11]. Yeast inocula were prepared as follows: 48 h prior to instillation in mice or seeding into tissue culture wells, fresh slants were started from the storage slants maintained for each yeast strain. After 24 h, new growth on slants was washed off into Sabdex broth, and incubated on a shaker overnight at 378C. On the day of the experiment, yeast were centrifuged for 15 min at 3000 g . The supernatant was discarded and the pellet was washed in sterile phosphate-buffered saline (PBS), resuspended, and centrifuged a second time. The pellet was then resuspended in a small volume of PBS, and an
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aliquot was counted in a hemacytometer. Yeast concentration was adjusted to give the desired concentration in the appropriate volume. For in-vitro experiments, yeast were also opsonized by addition of 0.25 vol. of normal mouse serum.

Cells
BV2 is a retrovirally-transformed murine microglial cell line developed and described by E. Blasi et al. [12]. The cells were the gift of D. J. Selkoe of Harvard Medical School. BV2 cells were maintained at 378C in a 5% CO2 atmosphere in RPMI 1640 medium (Gibco, Grand Island, NY, USA) supplemented by 200 mmol/l Lglutamine (Sigma, St Louis, MO, USA) N-[2-hydroxyethyl] piperazine-N?-[2-ethanesulfonic acid] (HEPES) buffer (Sigma), penicillin/streptomycin (Sigma), and 10% fetal bovine serum. T cells were obtained from the tracheobronchial lymph nodes of C57BL/6J mice that had been immunized 8 weeks previously by intratracheal instillation of a sublethal dose of strain 184 C. neoformans, and then boosted 4 days prior to the experiment by intranasal inhalation of 5 )/105 heat-killed strain 184 cryptococci or avirulent Ura5 cryptococci. Mice were killed by CO2 asphyxiation, and lymph nodes were removed aseptically and processed to obtain single cell suspensions. In experiments where T-cell subsets were purified, lymph node suspensions were first treated with FC block (anti CD16/CD32 clone 2.4G2, produced by the Trudeau Institute Monoclonal Antibody Facility), then incubated with fluorescein isothiocyanate (FITC)-antiCD4 (Pharmingen, San Diego, CA, USA) and Cy-chrome anti-CD8 (Pharmingen), then sorted by fluorescence activated cell sorter (FACStar, Becton Dickinson, Mountain View, CA, USA). Aliquots of T-cell suspension were routinely plated on Sabdex agar to verify that yeast derived from the vaccination or boost were either entirely absent or present in small numbers that were insignificant in comparison to the number of reporter strain organisms in the given experiment.

cutaneous layer only was penetrated. Intravenous challenge infections were delivered to the retro-orbital sinus via a 22 gauge needle. Typically under our rechallenge protocol, 2 )/104 organisms were injected in 200 ml fluid into the retro-orbital sinus. For the lowdose studies, concentration was adjusted to deliver the desired number of yeast in 200 ml.

Fungistasis assay
Twenty-four hours prior to addition of cryptococcal cells, 5 )/104 BV2 cells were added to wells of a 96-well tissue cultured treated culture dish, in supplemented RPMI 1640 medium plus 10% fetal bovine serum. When experimentally appropriate, cells received interferon (IFN)-g (final concentration, 100 U/ml) (X63IFN-g culture supernatant, a gift of L.L. Johnson, Trudeau Institute), and lipopolysaccharide (LPS) at 10 ng/ml. In some experiments, T-cell suspensions prepared as described above were added, also at 5 )/ 104 cells per well. Yeast cells opsonized by incubation in normal mouse serum for 30 min at room temperature were added to wells at a 20:1 effector (microglial cells): target (yeast cell) ratio. Control wells contained heatkilled (1 h, 568C) effector cells, and yeast cells. Yeast colony forming units (c.f.u.) per well were determined at time zero by plating the inoculum suspension on Sabdex agar; 96-well cultures were incubated for 48 h at 378C, 5% CO2. Individual conditions were tested in five wells per treatment. Total (extracellular and phagocytized) c.f.u. of yeast cells per well was determined by collecting supernatant fluid, lying the monolayer by addition of 0.2 ml sterile water to each well for 0.5 h (mammalian cells are lysed while C. neoformans cells remain intact and viable) and adding the resultant suspension to the appropriate supernatant sample already collected. Plates were visually inspected under the microscope to assure complete removal of cells from wells. Samples were centrifuged and resuspended in PBS, diluted and plated on sabdex agar as described [11]. Colonies were counted 48 hours later. Percent inhibition was calculated by the following formula. 100 (/(number of c.f.u. experimental) }/(number of c.f.u. 48 h control plate) )/100 0/%inhibition.

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Infections
Intratracheal C. neoformans infections were administered as previously described [13]. Briefly, mice were anesthetized by a mixture of halothane/oxygen and immobilized on an upright lucite board. A 1-ml syringe fitted with a blunted and bent 16 gauge needle was inserted in the top of the trachea, and 106 organisms were delivered in 100 ml sterile PBS. For subcutaneous infections [14], 106 organisms were delivered via a 22 gauge needle to a fold of skin between the inner thigh and the abdomen at a shallow angle, such that the
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Flow cytometry
BV2 microglial cells were stained for fluorescenceactivated cell sorter (FACSTM) analysis, utilizing antiMHC class I (H-2Db) (KH95) and anti-MHC class II (IAb) (KH29) antibodies from Pharmingen. 50 000 cells were acquired on a FACScan flow cytometer and the data were analyzed using CellQuest software (Becton

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Dickinson Immunocytometry Systems, San Jose, CA, USA). FACS sorting was performed on a FACStar Plus (Becton Dickinson). Pooled lymph nodes from five immune C57BL/6J mice were used in T cell plus microglial cell in vitro fungistasis assays. Antibodies to CD4 (GK1.5) and CD8 (TIB210) were produced by the Trudeau Institute monoclonal antibody facility. A lymphocyte gate was set and purified CD4 ' and CD8' T-cell subsets obtained, each at /99% purity.

Experimental strategy and results

Failure of antigen-experienced Ab null mice to resist intravenous infection with C. neoformans
In a previous study [5], we concluded that class-IIdeficient, CD4 ' T-cell-deficient Ab null mice could not be protected against C. neoformans brain infection by passive transfer of lymphocytes from wild-type immunized C57BL6/J donor mice. To investigate whether doubly class II-deficient and CD4' T-cell-deficient Ab null mice, whose entire development had occurred in the absence of CD4 ' T cells, could be directly vaccinated to generate resistance to lethal cryptococcal rechallenge, groups of mice were given an intratracheal instillation of 106 strain 184 C. neoformans organisms or of vehicle, under the protocols established in our laboratory for this immunization of mice [11,15]. Under these protocols, mice are rested for eight weeks post infection, in order to allow immunity to develop. After 8 weeks, both antigen-experienced and na ve mice are ordinarily challenged with an intravenous injection of 2 )/104 C. neoformans organisms of either an avirulent reporter strain (Ura5 ) or of strain 184. However, of 12 Ab null mice intratracheally infected during the vaccination protocol, none survived longer than five weeks post infection, but Ab null control mice that received vehicle all survived /80 days (Table 1). In several cases, moribund mice were euthanized, brains were harvested, and cryptococcal organisms were enumerated. Moribund mice were hydrocephalic and ataxic, and typically had greater than 2 )/108 cryptococci in their brains. A second immunization attempt had similar results. Nine Ab null mice were treated as described, and C57BL6/J mice received aliquots of the same cryptococcal inoculum, also intratracheally. Again, infected Ab null mice died between 3 and 6 weeks infection, but C57BL6/J and vehicle-treated mice survived (Table 1). From these

RNAse protection
IFN-g mRNA expression was analyzed by RNAse protection assay (RPA). Total RNA was harvested from 107 positively-sorted CD4' T cells and from an equivalent number of positively sorted CD8' T cells, using an RNeasy kit (Qiagen, Valencia, CA, USA). RiboQuant multiprobe RPA were performed according to the manufacturers (Pharmingen) instructions, to qualitatively assess cytokine mRNA expression levels. 32 P-labeled RNA probes (mCK-1 multiprobe template set) were hybridized to 5-mg samples of RNA. Samples were loaded onto sequencing gels and visualized using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA, USA).

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Statistical analyses
Numbers (log10) of viable C. neoformans cells are expressed as the means9/standard deviations (SD). Data were submitted to one-way statistical analysis of variance (ANOVA) with PRISM graphpad software (GraphPad Software, San Diego, CA, USA), with differences deemed significant at P B/0.05. Survival curves were analyzed via the Mann /Whitney test, again with differences deemed significant at P B/0.05.

Table 1 Lethality to Ab null mice of intratracheal vaccination with an ordinarily sub-lethal dose of strain 184 Cryptococcus neoformans Mice First vaccination attempt Ab null (n 0/12) Ab null (n 0/4) Second vaccination attempt Ab null (n 0/9) C57BL6/J (n 0/10) Ab null (n 0/4) C. neoformans dose 106 organisms/mouse vehicle given only 106 organisms/mouse 106 organisms/mouse vehicle given only Survival post intratracheal infection (days)

(20,20*,27,29,30*,31,31*,31*,32,33,34*,34) (all /80 days) (22,28,28,31,32,33,33,33,33,35) (all /70 days) (all /70 days)

*On a particular day, a mouse or mice were found to be moribund and were killed. Brains of asterisked mice were found to have between 2 and 4.3 )/108 c.f.u. strain 184 C. neoformans .

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results, we concluded that class-II-deficient, CD4' Tcell-deficient Ab null mice could not be immunized intratracheally under our ordinary laboratory protocol. Working with inducible nitric oxide synthase (iNOS)deficient knockout (iNOS KO) mice, we developed an alternative immunization protocol [14]. Under that protocol, mice that survived subcutaneous instillation of 106 strain 184 organisms for 8 weeks were found to have been rendered resistant to an ordinarily lethal intravenous re-challenge. We found that the majority of Ab null mice (12/14) also survived the subcutaneous vaccination protocol. Eight weeks post subcutaneous vaccination, five vaccinated Ab null mice and five na ve Ab null control mice were challenged with 2 )/104 avirulent Ura5 cryptococci. Use of the Ura5 strain allows us to distinguish, by differential plating, between the vaccinating and challenge doses. Relative resistance to yeast infection was measured by enumerating brain and lung Ura5 yeast burdens in mice killed 14 days post infection. Immunized mice had significantly fewer ura5 c.f.u. in both lungs and brains than did na ve controls (Fig. 1). Strain 184 organisms were not recovered from animals, suggesting that for the most part the vaccinating inoculum had been cleared, although we cannot entirely rule out the possibility that some organisms remained. In order to investigate whether subcutaneous immunization offered protection against an ordinarily

Fig. 2 Inuence of vaccination on survival times of vaccinated mice given a lethal intravenous cryptococcal challenge. Groups of 10 male Ab null mice were either vaccinated by subcutaneous instillation of 106 strain 184 cryptococci in 100 ml sterile PBS, or given a control injection of 100 ml PBS. All mice were rested 10 weeks, then given an intravenous rechallenge with 2 )/104 yeast organisms. Mice were allowed to progress to morbidity/mortality and dates of death were recorded. The two survival curves shown do not differ signicantly. The data shown is representative of two separate experiments.

lethal cryptococcal infection, 10 vaccinated Ab null mice and 10 na ve Ab null mice were challenged at 8 weeks post infection with intravenous infection of 2 )/104 strain 184 cryptococci. Mice were allowed to progress to morbidity or mortality. There was no difference between survival time of immunized mice and na ve controls (Fig. 2). From these data, we concluded that the protection seen in the experiments shown in Fig. 1 was transient and did not offer any extension of survival time in class-II-deficient, CD4' T-cell-deficient Ab null mice.

Effective vaccination of Ab null mice against a low-dose intravenous infection with C. neoformans
Reasoning that in AIDS patients and in other immunocompromized individuals, seeding of C. neoformans to the brain most likely occurs via sporadic, occasional release of organisms from the primary pulmonary foci to the vasculature, we decided to modify our protocol. A titration experiment was designed in order to find a challenge dose that was more physiologically relevant than the intravenous bolus of 2 )/104 virulent strain 184 yeast ordinarily delivered under our challenge protocol. Doses of 5 )/103, 103, and 5 )/102 organisms were delivered intravenously to groups of four vaccinated and four na ve Ab null mice, with the goal of finding an arguably physiologically relevant dose that extended survival of immune mice significantly beyond that of na ve controls. Immunized mice that were challenged by intravenous instillation of 1000 or fewer

Fig. 1 Effect of vaccination on yeast burdens in lower organs of Ab null mice at 12 days infection. Groups of ve male Ab null mice were either vaccinated by subcutaneous instillation of 106 strain 184 cryptococci in 100 ml sterile PBS, or given a control injection of 100 ml PBS. All mice were rested ten weeks, then given an intravenous rechallenge with 2 )/104 ura5 yeast organisms. At 12 days infection, mice were killed by CO2 asphyxiation, and brains and lungs were harvested. Organs were homogenized, serially diluted, and plated on Sabdex agar and YEPD, in order to differentially enumerate ura5 yeast colonies 48 h later. c.f.u. due to ura5 were signicantly higher (P B/0.05) in both lungs and brains of unvaccinated mice than in organs of vaccinated mice. Data shown are means9/standard deviation. Data are representative of two iterations of the experiment.

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Table 2 Survival times of vaccinated Ab null mice and na ve Ab null mice given a low-dose intravenous re-challenge Dose (per mouse) 2 )/10
4

Vaccinated or na ve Naive Vaccinated Na ve Vaccinated Na ve Vaccinated Na ve Vaccinated

Survival post intravenous infection (14,14,18,20) (15,15,17,22) (18,22,28,36) (14,26,29,30) (22,26,31,31) (34,37,39,50) (18,30,34,35) (54,56,75,81)

Significance / / Not different from na ve / / Not different from na ve / / Differs from naive / / Differs from na ve

5 )/103 103 2 )/102

Mice were vaccinated by subcutaneous instillation of 106 strain 184 cryptococci. After 8 weeks, mice were re-challenged, along with na ve controls, with varying doses of strain 184. Mice were set on the shelf and monitored for progression to morbidity/mortality. Significant differences were calculated by Mann /Whitney test, with significance assigned at P -values of B/0.05.

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organisms survived significantly longer than did na ve controls given the same intravenous challenge (Table 2). In our experience, retro-orbital sinus injection of 1000 organisms is likely to result in initial seeding of the brain with between 0.1 and 1.0% of the inoculum. Therefore, subcutaneous vaccination of mice could be seen to generate an immunity to low-dose re-challenge in CD4 ' T-cell-deficient Ab null mice.

that were CD8 positive, but mice that received the control antibody had between 1 and 10% of cells within

Diminished survival after low-dose infection with C. neoformans of Ab null mice depleted of CD8 ' T cells
Groups of na ve and vaccinated Ab null mice were treated with monoclonal antibody to CD8, or with an irrelevant isotype-matched control antibody, to assess the effect of CD8 ' T-cell depletion on the observed resistance to low-dose C. neoformans infection. Na ve mice treated with antibody to CD8 had significantly shorter survival time than did na ve mice that received the control antibody (Fig. 3). Similarly, vaccinated mice that received antibody to CD8 had a survival time significantly shorter than that of vaccinated mice that received control antibody (Fig. 3). Attempts to deplete lymphocyte subsets in long-term survival studies are often hampered by the mouses ability to mount an anti-antibody response that hastens clearance of the depleting antibody, or by the simple inability of experimenters to infuse enough antibody into the mouse to keep pace with its ability to regenerate lymphocytes. Although it was unlikely that these CD4' T-cell-deficient mice could mount a robust antibody defense in the absence of CD4 ' T-cellmediated help to B cells, we decided to assess the extent of CD8 ' T-cell depletion by flow cytometric analysis of brain-draining (deep cervical) lymph nodes of immune mice given a low-dose challenge followed by experimental and control mAb treatments. In all cases, mice that received monoclonal antibody versus CD8 had fewer than 1% of cells within the lymphocyte gate

Fig. 3 Effect of treatment with anti-CD 8 monoclonal antibodies on survival of Ab null mice given a low-dose rechallenge. Groups of 10 female Ab null mice were vaccinated or left na ve (as described for the experiment shown in Fig. 2). Ten weeks later, ve na ve and ve vaccinated Ab null mice were given 1 mg monoclonal antibody TIB210 vs CD8, and ve na ve and ve vaccinated Ab null controls were given 1 mg of an isotype matched control monoclonal raised against an irrelevant antigen; 24 h later, all mice received an intravenous challenge with 500 yeast organisms. Mice were given booster injections of 0.5 mg antibody as appropriate every 5 days. Mice were allowed to progress to morbidity/mortality and dates of death were recorded. Data were analyzed via Mann /Whitney test. For both na ve and immune mice, survival time was signicantly shorter for mice given mAb vs. CD8 than for mice given control antibody (P B/0.05). The data shown are representative of two separate experiments.

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the lymphocyte gate that were CD8 positive. CD4 ' T lymphocytes were absent or present at less than 1% of cells within the lymphocyte gate in both treatment groups (data not shown). Taken together, the previous data demonstrate a resistance to low-dose C. neoformans brain infection in the absence of CD4 ' T cells, that is at least partially CD8' T-cell-dependent.

Relative efficiencies of CD4' and CD8' T cells in providing stimulatory signals to BV2 cells in fungistasis assays
To investigate a possible mechanism for the observed inferiority of CD8' T-cell-mediated resistance to C. neoformans brain infection, interactions of T-cell subsets with brain-derived macrophages were examined in vitro. BV2 is a retrovirally transformed mouse microglial cell line [12] that has the capability to exert an antimicrobial effect against C. neoformans [16,17]. In our hands, IFN-g-stimulated (100 U/ml) BV2 cells were efficient inhibitors of proliferation of strains 145, cap 67, 184, and 52D (data not shown). IFN-g, while primarily a T-cell-derived cytokine, can also be secreted by other cell types. To determine whether lymphocytes could deliver effective stimulatory signals to BV2 cells, the microglia were co-cultured with equivalent numbers (5 )/104 per well) of nylon-wool non-adherent cells harvested from lymph nodes. Cells were obtained as follows. Immune C57BL6/J mice were given a secondary exposure to C. neoformans by intranasal instillation of avirulent ura5 organisms. Four days later, lymph nodes draining the lung were harvested and a single cell suspension was prepared. To test whether CD4 ' T cells and CD8 ' T cells were equally efficient at stimulating BV2 cells to fungistasis, lymphocyte suspensions from immune and boosted mice collected as described above were labeled with antibody to CD4 or CD8 molecule, and sorted by flow cytometry to give two separate preparations of purified CD4' or CD8 ' positive cells. Cell concentration was adjusted to 5 )/105 per ml and 100 ml of suspension was delivered per well. 5 )/104 CD4' T cells or 5 )/104 CD8' T cells were added separately or together to wells along with BV2 cells. Four replicate wells were set up for each treatment group. The treatment groups were as follows. (i) IFN-g-stimulated BV2 cells, (ii) medium only and BV2 cells, (iii) a mixed population of CD4' cells and CD8' cells and BV2 cells, (iv) CD8' cells and BV2 cells, and (v) CD4 ' T cells and BV2 cells. All wells received strain 184 C. neoformans cells at a 20:1 effector-to-target (E:T) ratio, in this case a BV2:yeast ratio. After 24 h, wells were harvested and supernatant and cell lysates combined and plated for
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yeast enumeration. Results were reported as percent inhibition of yeast cell growth compared to growth in control wells that contained only heat-killed BV2 cells and yeast cells. CD4 ' T cells efficiently stimulated BV2 cells to inhibit yeast proliferation (Fig. 4). Moreover, at the concentration tested, CD4' cells appeared to deliver stimulatory signals to microglia as potent as those delivered by exogenous addition of IFN-g. BV2 cells incubated with CD8' T lymphocytes inhibited yeast proliferation considerably less well than did CD4' T cells. It should be noted that this assay was performed four times with varying results. In two experiments, wells with BV2 cells and CD8' T cells did not have significantly fewer yeast cells than did wells with heat-killed BV2 cells only, although a trend toward fewer yeast cells was observed. In two experiments, significant CD8' T-cell-mediated fungistasis occurred, albeit at a lower rate than in wells where BV2 cells were co-cultivated with CD4 ' cells. The experiment shown in Fig. 4 is representative of the two experiments where CD8' T cells did succeed in significantly inhibiting yeast proliferation. From these data, we confidently concluded that CD4' T cells are more efficient stimulators of BV2 anti-cryptococcal

Fig. 4 CD4' T-cell- vs. CD8 ' T-cell-mediated potentiation of microglial fungistasis. Twenty-four hours prior to addition of cryptococal cells (strain 184, 2500 organisms), 5 )/104 BV2 cells were added to wells of a 96-well tissue culture treated dish, along with (A) IFN-g (100 U/ml), (B) medium only, (C) 5 )/104 nylon wool nonadherent cells harvested from lymph nodes of antigen-experienced and boosted B6 mice (see Materials and methods), (D) 5 )/104 puried CD8 ' T cells prepared as described in C, or (E) 5 )/104 puried CD4 ' T cells prepared as described in C. Data shown are mean percent inhibition9/standard deviation, with four wells per treatment group. Note that these data are representative of two experiments in which co-culture with CD8 ' T cells resulted in statistically signicant inhibition of yeast proliferation (see text for details). *Signicant compared to % inhibition seen in BV2 cells, no stimulation (B) (P B/0.05).

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activity than are CD8' cells, but that CD8 ' T cells can exhibit anti-cryptococcal activity. To test whether IFN-g expression differed between CD4' and CD8' T lymphocytes, aliquots from these purified antigen-experienced lymphocyte suspensions were held aside and processed for determination of IFN-g mRNA levels by RNAse protection. Both CD4' and CD8' cells produced measurable IFN-g mRNA, but densitometric analysis revealed no significant difference in levels of the cytokine between the two samples (data not shown).

Constitutive and inducible expression of MHC antigens by BV2 cells

Microglial cells have been reported to constitutively express both MHC class I and class II antigen. Upon stimulation with IFN-g, microglia are reported to upregulate MHC class II expression, which would allow them to interact with CD4' T cells [18,19]. In order to characterize expression of the major histocompatibility antigens in this particular transformed cell line (BV2), we incubated cells for 24 h in the presence and absence of IFN-g, then harvested them and labeled them with antibodies to MHC class I and MHC class II. We then analyzed them by flow cytometry. In the absence of IFN-g, BV2 cells constitutively expressed MHC I and MHC II antigen. In the presence of IFN-g, the prominent CY5-stained MHC I peak shifted from about 2.2 to 2.8 logs (an approximately fourfold increase), but the FITC-stained MHC II peak shifted from 1.1 to 2.25 logs (an approximately 14-fold increase) (Fig. 5). Therefore, IFN-g-inducible MHC class II expression may result in many more interactions of an immunologically productive nature between microglial cells and CD4' T cells than are stimulated between microglial cells and CD8' T cells by changes in MHC class I expression.

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Fig. 5 Regulation of MHC Class II and Class I antigen levels in response to addition of IFN-g. BV2 cells were cultivated at 378C and 5% CO2 for 24 h in the presence (heavy line) or absence (light line) of 100 U/ml IFN-g. Cells were harvested and stained with uorescenttagged antibody to MHC class II or class I molecule (see Materials and methods) and ow cytometric analyses were performed.

Discussion
Attempts to develop prophylactic and therapeutic treatments to protect at-risk populations from lethal cryptococcal meningoencephalitis have been hindered by the fact that anticryptococcal resistance has been observed only when infected animals possessed a healthy, functional CD4 ' T-cell compartment. CD4' T cells are likely to be the very subset most grievously compromised in AIDS patients, and they are also unlikely to be optimally functional in recipients of radiation therapy. Delineation of a protective role for CD8' T cells would encourage research into the rational design of a vaccine engineered to elicit CD8'

T cell memory. This laboratory began investigating anticryptococcal resistance in the class-II-deficient, CD4' T-cell-deficient Ab null mouse to explore the eventuality that a plasticity of lymphocyte function might allow CD8' T cells produced and matured in the absence of CD4 ' T cells to undertake some of the functions normally mediated by CD4' cells. Alternatively, it was thought, even a relatively modest anticryptococcal function inherent in CD8' T cells might be more easily detectable when CD4' cells were eliminated. At low doses of cryptococci, which we believe to be physiologically relevant, and in the absence of CD4' cells, CD8' T cells did indeed play a protective role, both in primary and re-challenge intravenous infections, both in the lungs and in the brain. CD8 ' T cells are a necessary component, along with CD4' cells, for containment and clearance of lung infection [13,20], but any contribution they made to
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anticryptococcal resistance in the brain had heretofore been undetectable [3,4]. The differences in help rendered by CD4 ' and CD8 ' T cells to anticryptococcal effectors, that is, to alveolar macrophages or microglial cells, might be qualitative or quantitative. CD8' T cells are recoverable from the brains as well as the lungs of experimentally infected mice (Aguirre, unpublished data), but it is not known whether their entry or retention at these two anatomical sites is equivalent. Nor is it known if the mechanisms of resistance differ qualitatively or quantitatively at the two anatomical sites. As is often the case in experiments done in vivo , details of the actual mechanisms causing death in lethally infected mice are at best only inferred. Mice lacking CD4' T cells were seen, in the present experiments, to die with profound hydrocephaly and ataxia. In addition, those mice for which examination was possible could be seen to have /108 yeast cells per brain, suggesting that they died of acute brain infection. Lung burdens were considerably lower at B/106 per animal (data not shown), a level that is generally well-tolerated in mice of this genetic background. The mechanism by which brain-derived macrophages (microglia) inhibit yeast proliferation is largely undefined. Microglia, and other macrophage-like cells, phagocytize cryptococci, but the fate of the ingested organisms is not always death. Studies in vitro with human microglia have shown that effective stasis of the invaders correlates with the presence of tight-fitting rather than loose phagosomes [21]. It has been suggested that inclusion of T lymphocytes in the microglial cell cultures might have potentiated anticryptococcal activity. Cryptococci have also been reported to inactivate macrophages by fostering the accumulation of cytoplasmic vesicles filled with intracellular polysaccharide [22] that then tends to become associated with potentially cytotoxic rod-like crystalline structures in the cytoplasm [23]. Macrophages need not even ingest the fungi, if an important mechanism of action involves generation of NO or reactive oxygen intermediates (ROI) that may cross cellular membranes and act extracellularly. INOS is a necessary component of anticryptococcal resistance, at least in primary C. neoformans infection, although activity of this inducible NO-synthesizing enzyme is not required for clearance of secondary re-challenge infections in immune animals [14]. The in-vitro assay system described in the present study may be an excellent tool for discovery of differential gene expression in microglia in response to T-cell potentiation, and may therefore ultimately lead to elucidation of an important mechanism for the elimination of invading microbes in the CNS.
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The immune function of microglial cells is as yet not well defined. We found that a brain-derived, macrophage-like transformed microglial cell line, BV2, was capable of responding to potentiating signals delivered by T cells of the CD4' subset to significantly inhibit the proliferation of C. neoformans in each of four assays. We also show that CD8' T-cell potentiation was less robust, achieving statistical significance in only two out of four assays, although a trend toward inhibition of yeast proliferation was observed in all assays. When BV2 cells were stimulated with unfractionated lymphocyte suspensions, inhibition of proliferation was comparable to that seen with purified CD4' T cells, arguing against an actual inhibitory effect of CD8' T cells in this system. It is interesting to note that we have consistently observed fungistasis in properly stimulated microglial cultures, but we have only rarely observed a fungicidal effect. Only in one or two of many experiments did we ever observe instances where fewer yeast cells were retrieved from a microglial culture than were originally seeded into it. These instances were in cultures with E:T ratios of at least 100:1 (data not shown). As we have observed clearance of large numbers of yeasts from brains of mice infected experimentally in vivo [15], it seems unlikely that such a large E:T ratio is necessary for fungal killing in a physiological setting. It is likely that not all the components that are present at foci of infection are present in our culture assay system. It should also, however, be noted that natural infection of the CNS in AIDS patients or other immunocompromized individuals is likely to occur in a random, stochastic fashion. Relatively infrequent events occur in which individual organisms or small clusters breach the pulmonary defenses and break into the vasculature to seed distant organs, including the brain. Therefore, E:T ratios of far greater than 100:1 may often occur, at least in the early stages of cryptococcal brain infection. In the present study, we utilized putatively physiologically relevant levels of brain seeding in our low-dose experiments. In these experiments, CD8 ' T cells did indeed play a protective role, both in primary and rechallenge intravenous infections, since depletion of CD8' cells resulted in loss of resistance. The in vitro fungistasis experiments demonstrated that CD4 ' and CD8' T lymphocytes differed in the efficacy with which they provided stimulatory help to microglial effectors. Co-cultures of BV2 cells and CD4' T cells incubated with no addition of stimulatory cytokines or factors exhibited more efficient fungistasis than was seen with co-cultures of BV2 cells and CD8' T cells. IFNg has been repeatedly identified as an essential

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component of anticryptococcal resistance mechanisms [1,24 /29]. It induces up-regulation of MHC class II molecules on other macrophage-derived cells. Our data (Fig. 5) suggest that BV2 cells express a constitutive, low but detectable level of class II molecule that is dramatically up-regulated in the presence of IFN-g. On the other hand, class I molecule in the same system is largely insensitive to the presence of the cytokine. While the corresponding increases in mean fluorescence intensity seen in flow cytometry do not give us information about the absolute numbers and relative densities of class I and class II molecules on microglia, they are suggestive of an enhancement of microglial interaction with CD4 ' T cells in the course of inflammatory events, an enhancement that is less evident for interactions with CD8 ' cells. A given cytokine-stimulated microglial cell may be less likely to form an interaction with a CD8' cell than with a CD4' cell. Even if it is tentatively assumed that natural killer (NK) cell-derived IFN-g might be present to initiate the immune potentiation of the host defense cells present at a lesion, it nonetheless remains likely that a continuous level or even an increased local concentration of IFN-g is necessary to maintain and propagate a strong inflammatory reaction. Perhaps coordinated interactions of CD4 ' T cells and microglia ensure that high local concentrations of the cytokine are focused on the microglial cell surface. Or perhaps rounds of clonal T-cell proliferation, dependent upon concerted T-cell/APC action at the site of the lesion, are required for optimal propagation and continued maintenance of the local immune response. In either case, CD4' T-cell-mediated responses are likely to be more robust than CD8 ' T-cell-mediated events, such as cytotoxicity or cytokine secretion carried out by CD8' T cells. We found that CD8 ' T cells can play a protective role against C. neoformans brain infection, provided infection occurs a few organisms at a time, rather than as a large, physiologically unrealistic experimental bolus. Our finding of an apparent molecular basis for the relatively weak CD8 ' T-cell response as compared to that delivered by CD4 ' T cells contributes significantly to the understanding of interactions between lymphocytes and brain-derived macrophages. Additionally, our novel modification extending previously described in-vitro fungistasis assays to include the contribution of co-cultured antigen-experienced T lymphocytes provides a system that may be used for further investigation of the dynamic interaction of microbes, innate host defense cells and lymphocytes in CNS infection.

Acknowledgements
This work was supported by grant AI418881 of the National Institutes of Health, and by institutional support from Trudeau Institute and Clarkson University.

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