INFECTION AND IMMUNITY, July 2010, p. 3097–3102 Vol. 78, No. 70019-9567/10/$12.00 doi:10.1128/IAI.00181-10Copyright © 2010, American Society for Microbiology. All Rights Reserved.

Optimal CD8 T-Cell Response against Encephalitozoon cuniculi IsMediated by Toll-Like Receptor 4 Upregulation

by Dendritic Cells�

Elizabeth M. Lawlor, Magali M. Moretto, and Imtiaz A. Khan*Department of Microbiology, Immunology and Tropical Medicine, George Washington University, Washington, DC 20037

Received 22 February 2010/Returned for modification 4 April 2010/Accepted 18 April 2010

CD8� T-cell immunity has been shown to play an important role in the protective immune response againstEncephalitozoon cuniculi. Although earlier studies suggest that dendritic cells (DC) are important for theinduction of this response, the factors responsible for initiation of the dendritic cell response against thispathogen have not been evaluated. In the current study, we demonstrate that E. cuniculi infection causes strongToll-like receptor 4 (TLR4)-dependent dendritic cell activation and a blockade of this molecule reduces theability of DC to prime an antigen-specific CD8� T-cell response. Pretreatment of DC with anti-TLR4 antibodycauses a defect in both in vitro and in vivo CD8� T-cell priming. These findings, for the first time, emphasizethe contribution of TLR4 in the induction of CD8� T-cell immunity against E. cuniculi infection.

Microsporidia are small obligate intracellular parasites that,until recently, were thought to be protozoans; however, evi-dence now suggests that they are related to fungi (15, 17).Microsporidia can infect a vast number of species of verte-brates and invertebrates; of the 150 genera of microsporidia,however, only 7 have been found to infect humans (13). Severeinfections have been reported predominantly for immunocom-promised patients, such as patients with HIV and organ trans-plant recipients (2, 7, 23, 37). Acute infections have also beenreported in travelers and the elderly (26, 27), and there isevidence of colonization of healthy, nonsymptomatic patients(34).

Due to the prevalence of opportunistic microsporidian in-fections associated with the HIV-AIDS pandemic, recent re-search has focused on the host’s immune response to thesepathogens. Early animal studies showed that cellular immunitywas necessary to protect SCID mice from a lethal Encephali-tozoon cuniculi challenge. Moreover, depletion of CD8� Tcells caused mice to succumb to intraperitoneal (i.p.) E. cunic-uli infection (21), and previous studies in our laboratory haveshown that cytotoxic lymphocytes play a major role in protec-tion against this effect (20, 21).

Recent reports from our laboratory have demonstrated thatdendritic cells (DC) play an important role in the priming ofthe immune response against E. cuniculi (31, 32). T cells incu-bated with E. cuniculi-pulsed DC exhibited antigen-specificcharacteristics, specifically gamma interferon (IFN-�) produc-tion, cytotoxicity, and proliferation (31, 32). In order to mountan immune response against a foreign pathogen, DC must firstrecognize the pathogen to initiate an appropriate response.One key method of recognition is through Toll-like receptors(TLRs), which were first discovered in Drosophila in response

to infection with fungal pathogens (24). However, specific TLRmolecules involved in DC activation during E. cuniculi infec-tion have not been identified previously. We evaluated theupregulation of specific molecules involved in activation of theDC response after E. cuniculi infection. Different TLR mole-cules were tested, and TLR4 expression was found to be es-sential for induction of the optimal CD8� T-cell response bythese cells.

MATERIALS AND METHODS

Mice. Six- to 8-week-old C57BL/6 mice were purchased from the NationalCancer Institute (Frederick, MD). The animals were housed under InstitutionalAnimal Care and Use Committee-approved conditions at the Animal ResearchFacility at The George Washington University (Washington, DC).

Parasites and infection. A rabbit isolate of E. cuniculi (genotype II), kindlyprovided by L. Weiss (Albert Einstein College of Medicine, Bronx, NY), wasused throughout this study. The parasites were maintained by continuous pas-sage in rabbit kidney (RK-13) cells, obtained from the American Type CultureCollection (Manassas, VA). The RK-13 cells were maintained in RPMI 1640containing 10% fetal calf serum (FCS) (HyClone Laboratories, Logan, UT).Mice were infected via the intraperitoneal (i.p.) route with 2 � 107 spores/mouse.In vitro stimulation was performed using irradiated parasites (220 krads).

TLR expression by dendritic cells. Expression of TLR2, -4, and -9 by dendriticcells was assessed on various days postinfection (p.i.) (2, 4, and 6 p.i.) by per-forming a phenotypic analysis. Briefly, spleens were harvested, and this wasfollowed by enzymatic (collagenase D and DNase I) and mechanical, disruption,allowing for DC separation. The cell suspension was labeled for CD11c, NK1.1,CD19, and TLR2 (eBioscience, San Diego CA) or TLR4 (BD Bioscience, SanJose CA) expression. Intracellular TLR9 expression was determined after per-meabilization and fixation with FoxP3 staining buffer (eBioscience) and intra-cellular staining with anti-TLR9 antibody (eBioscience). Cells were acquiredwith a FACSCalibur (BD Biosciences) and were analyzed with FlowJo (TreeStar, Inc., Ashland, OR).

TLR2, -4, and -9 messages were detected by real-time PCR according tostandard protocol in our laboratory (45). Splenic DC were isolated according toa previously described protocol (45). Briefly, spleens were harvested as describedabove. A cell suspension was then labeled with anti-CD11c biotin-conjugatedantibodies (eBioscience) and positively selected by magnetic purification usingthe manufacturer’s protocol (Stem Cell Technology, Vancouver, British Colum-bia, Canada). Positively selected cells were then labeled, and CD11c� CD19�

NK1.1� DC were purified using a cell sorter (FACSAria; BD Biosciences). RNAwas isolated with an RNeasy kit (Qiagen, Valencia, CA) according to the man-ufacturer’s instructions and reverse transcribed with Moloney murine leukemiavirus (MMLV) reverse transcriptase (Invitrogen, Carlsbad, CA). Quantitative

* Corresponding author. Mailing address: Department of Microbi-ology, Tropical Medicine and Immunology, George Washington Uni-versity, 2300 I Street, Washington, DC 20037. Phone: (202) 994-2863.Fax: (202) 994-2913. E-mail: mtmixk@gwumc.edu.

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real-time PCR was carried out using an MyiQ single-color real-time PCR detec-tion system (Bio-Rad, Hercules CA) and each of the following primers at a concen-tration of 200 nM: 5mTLR2 (5�-GCCACCATTTCCACGGACT-3�), 3mTLR2 (5�-ACCTGGCTGGTTTACACGTC-3�), 5mTLR4 (5�-GTGCCAGAGACATTGCAGAA-3�), 3mTLR4 (5�-GGCTTCCTCTTGGCCTGG-3�), 5mTLR9 (5�-ACTGAGCACCCCTGCTTCTA-3�), 3mTLR9 (5�-AGATTAGTCAGCGGCAGGAA-3�),5m�-actin (5�-AGAGGGAAATCGTGCGTGAC-3�), and 3m�-actin (5�-CAATTAGTGATGACCTGGCCGT-3�). Amplification was performed with SYBRGreenER qPCR Supermix (Invitrogen) for TLR2 and TLR4 or with iQ SYBRGreen Supermix (Bio-Rad) for TLR9 and consisted of 40 cycles of 95°C for 15 s and60.5°C for 1 min, followed by melt curve analysis. A baseline value of 1.0 wasestablished using DC from uninfected mice.

Detection of intracellular IL-12. To assess interleukin-12 (IL-12) production,splenic DC were isolated as described above. DC were plated at a concentrationof 5 � 105 DC/well and incubated with monensin (BD Biosciences) overnight.On the following day, cells were stained for surface markers using anti-CD11c,anti-NK1.1, and anti-CD19 antibodies. The cells were then fixed and permeab-ilized using a Cytofix/Cytoperm kit (BD Bioscience) according to the manufac-turer’s instructions and were labeled with anti-IL-12p40 antibody (eBioscience).

Immunoprecipitation. Splenic dendritic cells from naïve and day 4 infectedmice were isolated as described above. Purified DC were then incubated withlysis buffer (20 mM Tris-HCl, 137 mM NaCl, 10% glycerol, 1% NP-40, 2 mMEDTA) for 20 min on ice. Supernatants were collected following centrifugationat 12,000 rpm for 10 min and incubated with myeloid differentiating factor 88(MyD88) F-19 antibody (Santa Cruz Biotechnology, Santa Cruz CA) for 1 h andthen overnight with protein A/G agarose beads (Santa Cruz Biotechnology). Theimmunoprecipitated complex was separated on a precast 4 to 15% Tris-HCl geland transferred to a polyvinylidene difluoride membrane (Bio-Rad). After over-night incubation with 5% dry milk in TBST (Tris-NaCl with 0.05% Tween 20),membranes were incubated with anti-mouse TLR4 antibody (1:4,000; eBio-science) for 1 h and then with anti-rabbit IgG peroxidase-conjugated secondaryantibody (1:160,000; Sigma Aldrich, St. Louis, MO). The hybridized bands werevisualized by chemiluminescence using an ECL kit (Pierce).

Expression of costimulatory molecules. DC were purified as described aboveand plated at a concentration of 5 � 104 cells/well in a 96-well plate. The cellswere then incubated with 40 �g/ml of anti-TLR4 (MD-2) antibody (BD Bio-sciences) or rat IgG2a isotype control antibody (eBioscience) for 1.5 h beforeirradiated E. cuniculi was added (2.5 � 105 cells/wells). Following overnightincubation, cells were harvested and labeled with phycoerythrin (PE)-conjugatedanti-CD80, -CD86, or -major histocompatibility complex (MHC) class II anti-bodies (eBioscience). Data were acquired with a FACSAria (BD Biosciences)and were analyzed with FlowJo software.

Measurement of CD8� T-cell response. DC were isolated, treated with anti-TLR4 antibody, and incubated with irradiated E. cuniculi as described above.The next day, splenic TCR�� T cells were isolated from naïve mice and addedto the culture (5 � 105 cells/well). After 96 h of incubation, CD8� T-cellactivation was examined using Ki67 and IFN-� intracellular staining. Briefly,monensin was added to the culture overnight according to the manufacturer’sprotocol. Labeling of cells with anti-CD8� and anti-CD25 was followed bypermeabilization using a Cytofix/Cytoperm kit (BD Biosciences). Cells were thenlabeled with anti-IFN-� and anti-Ki67. Cells were acquired with a FACSCaliburand analyzed with Flowjo software.

Adoptive transfer of E. cuniculi-pulsed DC. DC were isolated, treated withanti-TLR4 antibody, and incubated with irradiated E. cuniculi as describedabove. The next day, 4 � 105 to 5 � 105 cells were adoptively transferred to naïveC57BL/6 mice via injection into the tail vein. At day 6 posttransfer, spleens wereharvested, and cell suspensions were prepared and incubated overnight withirradiated E. cuniculi spores in the presence of monensin. The next morning, cellswere labeled for CD8 and CD25 expression. After permeabilization as describedabove, cells were labeled with anti-IFN-� and -KI67 antibodies. Cells wereacquired with a FACSCalibur and were analyzed with Flowjo software.

Statistical analysis. Statistical analysis of the data was performed using atwo-tailed Student t test (33).

RESULTS

Splenic dendritic cell response following E. cuniculi infec-tion. DC have been shown to be the main antigen-presentingcells during infection (1) and can produce IL-12 in response tovarious pathogens (25). Previous studies from our laboratoryhave reported that aging mice with a defect in DC function are

unable to prime an optimal T-cell immunity against E. cuniculiinfection (31). Splenic DC were analyzed at different timepoints after E. cuniculi infection (days 2, 4, and 6 p.i.), andIL-12 expression was assessed by intracellular staining. Thefrequency of total DC as well as IL-12-producing DC peaked atday 4 p.i. and started to decline by day 6 (Fig. 1A to C). Theearly increase in the frequency of IL-12-producing DC in re-sponse to E. cuniculi infection demonstrates that these cellsplay an important role in initiation of the immune responseagainst E. cuniculi.

E. cuniculi infection induces TLR4 expression by splenicDC. DC are known to initiate an immune response throughrecognition of pathogen-associated molecular patterns (PAMPS)by Toll-like receptors. Microsporidia consist of a chitinousspore wall that contains O-linked glycans similar in structure tothose found in the cell wall of Candida albicans (38), which hasbeen shown to signal through TLR2 and -4 (3, 14). Addition-ally, microsporidia contain glycosylphosphatidylinositol (GPI)anchors analogous to those found in Trypanosome cruzi, Plas-modium falciparum, and Toxoplasma gondii, all of which ac-tivate TLR2 and/or TLR4 (4, 8, 22, 28). TLR2, -4, and -9expression was measured to determine if these molecules areinvolved in activation of DC during E. cuniculi infection.Spleens were harvested from infected animals at day 4 p.i., DCwere purified, and the levels of mRNA transcripts for TLR2,-4, and -9 were determined. As shown in Fig. 2A, significantupregulation of the TLR4 message was observed at day 4 p.i.Conversely, no significant increases in the messages for othertwo TLRs (TLR2 and TLR9) in response to E. cuniculi infec-

FIG. 1. Splenic DC response after E. cuniculi infection. C57BL/6mice were infected i.p. with 2 � 107 E. cuniculi spores. At differenttime points p.i. (days 2, 4, and 6), animals were sacrificed, spleens wereharvested, and single-cell suspensions were prepared by using collage-nase D and DNase I digestion. Cells were directly labeled for CD11c,CD19, and NK1.1 (A) or were incubated overnight with monensinbefore intracellular IL-12 staining (B and C). Experiments were per-formed twice, and the data are representative of the results of oneexperiment.

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tion were noted (Fig. 2A). To further determine the TLRmolecules involved in DC activation, the expression of thesemolecules by DC was measured by a flow cytometry assay. Asshown in Fig. 2B, there was no change in the mean fluores-cence intensity (MFI) for either TLR2 or TLR9 followinginfection. However, a significant increase in the TLR4 MFI atday 4 p.i. (P � 0.0001) was noted. These observations suggestthat splenic DC upregulate TLR4 in response to E. cuniculiinfection.

TLR4 expression is associated with MyD88. Since activationof TLR4 by lipopolysaccharide (LPS) stimulation has been shownto act in both myeloid differentiating factor 88 (MyD88)-depen-dent and MyD88-independent pathways (12, 19), we sought todetermine the involvement of MyD88 during E. cuniculi infection.Splenic DC were isolated at day 4 p.i., and the cell lysate wasimmunoprecipitated with anti-MyD88 antibody and subsequentlyimmunoblotted with anti-TLR4 antibody. As shown in Fig. 2C,DC from infected mice exhibited an increase in TLR4 proteininteracting with MyD88 (Fig. 2C), suggesting that TLR4 signalingin response to E. cuniculi infection is MyD88 dependent.

In vitro TLR4 blockade reduces the expression of costimu-latory molecules on DC. Upregulation of costimulatory mole-cules and antigen presentation by DC are essential for induc-tion of the optimal T-cell response (5). To further assess therole of TLR4 in the DC function, an in vitro blocking assay wasperformed. Splenic DC from naïve mice were isolated andincubated with anti-TLR4 antibody or an isotype control for1.5 h and then pulsed overnight with E. cuniculi spores. Sub-sequently, the cells were harvested, and the expression of co-stimulatory molecules (CD80, CD86, and MHC class II) was

measured by flow cytometry. As shown in Fig. 3A, blockingTLR4 prior to E. cuniculi stimulation prevented upregulationof all of the costimulatory molecules mentioned above. Toensure that the stimulation through TLR4 was not due to LPScontamination, polymyxin B (an antibiotic that binds to andneutralizes LPS) was added to DC cultures prior to the E.cuniculi pulse. Addition of polymyxin B to the cultures did notalter the expression of costimulatory molecules, ruling out thepossibility that LPS was involved (data not shown). Theseresults demonstrate that TLR4 blockade prevents upregulationof costimulatory molecules (CD80, CD86, and MHC class II)on DC, which most likely impairs their functions.

Reduced IL-12 production by DC after anti-TLR4 treat-ment. DC are known to be an important source of IL-12, acytokine reported to play an important role in the immuneresponse against E. cuniculi (35). Moreover, data shown in Fig.1 demonstrate that there was an increase in the frequency ofIL-12-producing DC in response to E. cuniculi infection. Next,we determined the effect of TLR4 blockade on IL-12 produc-tion by DC. Splenic DC were purified and treated with anti-mouse TLR4 antibody or an isotype control, as described

FIG. 2. TLR4 upregulation after E. cuniculi infection is MyD88dependent. Splenic DC were isolated at day 4 p.i. and analyzed forTLR2, TLR4, and TLR9 expression. TLR mRNA expression wasnormalized using �-actin mRNA levels (A). Relative expression wasmeasured using the mean for each group and the formula for therelative transcript level: 2�CT � 1,000 where CT is the cycle threshold.DC were labeled with CD11c, CD19, NK1.1, and either TLR2, TLR4,or TLR9 antibodies (B). Splenic DC were lysed and immunoprecipi-tated with anti-MyD88, and the complex was then immunoblotted forTLR4 (C). Lane MW contains a protein ladder. Experiments wereperformed twice, and the data are representative of two experiments.

FIG. 3. Costimulatory molecule expression and IL-12 productionare TLR4 dependent. Splenic DC were purified from naïve mice,plated, and incubated with anti-TLR4 (TLR4) or isotype controlantibody. Cells were stimulated overnight with irradiated E. cuniculiand stained for CD80, CD86, or MHC class II expression (A). Un-stimulated cells are represented by the shaded histogram. The meanfluorescence intensity is presented next to each antibody treatment.IL-12 production by splenic DC was measured by intracellular stainingafter overnight treatment with monensin (B and C). Experiments werecarried out twice, and the data are representative of one experiment.

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above. Cells were pulsed with E. cuniculi overnight, and IL-12was detected by intracellular staining the next day. As shown inFig. 3B, the frequency of IL-12� DC was significantly reducedafter anti-TLR4 treatment compared to the results for cellstreated with the isotype control (P � 0.006).

Inhibition of TLR4 signaling suppresses effective T-cellpriming. The data presented above demonstrate that TLR4blockade affects the upregulation of costimulatory moleculesknown to be critical for T-cell priming (5). To study the down-stream effect of this blockade, we determined if prior treat-ment with anti-TLR4 antibody inhibits the ability of DC toinitiate an optimal CD8� T-cell response against E. cuniculiinfection. Purified DC were isolated and treated with anti-mouse TLR4 or isotype control antibody, as described above.Following overnight stimulation with E. cuniculi spores, T cellswere added to the cultures, and 96 h later, the response wasmeasured by measuring IFN-�, CD25, and Ki67 expression. Asexpected, an increase in the frequency of CD25� IFN-��

CD8� T cells was observed when DC were incubated with the

isotype control (Fig. 4A). Conversely, prior treatment withTLR4 antibody significantly reduced the ability of these cells toprime the CD8� T-cell response compared to that of cellstreated with the isotype control (P � 0.0085) (Fig. 4A). Simi-larly, there was no significant increase in the percentage ofCD8� T cells (CD25� Ki67�) when DC were treated withantibody to TLR4. These observations suggest that this mole-cule is important for priming the CD8� T-cell immunityagainst E. cuniculi.

To further establish the importance of TLR4 signaling in thepriming of the CD8� T-cell response, adoptive transfer studieswere performed. Anti-TLR4 antibody-treated DC were pulsedwith E. cuniculi spores in vitro and subsequently transferred tonaïve syngeneic mice. At day 6 posttransfer, the recipient an-imals were sacrificed, and splenic CD8 T cells were evaluatedfor IFN-�, CD25, and KI67 expression. As shown in Fig. 4B,recipient animals that received anti-TLR4 antibody-treatedDC exhibited reduced T-cell priming compared to control an-imals, which received control antibody-treated DC. A signifi-

FIG. 4. Downregulated CD8� T-cell response after TLR4 blockade. Splenic DC purified from naïve mice were plated, incubated with eitheranti-TLR4 antibody or anti-rat IgG2a antibody, and pulsed overnight with irradiated E. cuniculi. For the in vitro assay, the next day, purified splenicCD8�� T cells were added to cultures (A). After 96 h of incubation, cells were harvested and labeled for CD8�, Ki67, IFN-�, and CD25. For thein vivo assay, E. cuniculi-pulsed DC treated with anti-TLR4 were adoptively transferred intravenously to naïve recipients (B). Six days posttransfer,spleens were harvested, and single-cell suspensions were prepared. After overnight incubation with monensin, cells were labeled for CD8�, KI67,IFN-�, and CD25. Cells displayed in all dot plots were gated on CD25� cells. Experiments were performed twice, and the data are representativeof the results of one experiment. NS, not significant.

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cantly lower percentage of IFN-�-producing cells was observedfor the CD25� CD8� T-cell population of these animals (P �0.0083). Similarly, the frequency of proliferating CD25� CD8�

T cells was significantly reduced in the mice that receivedanti-TLR4 antibody-treated DC (P � 0.00647) (Fig. 4B).These findings further establish that TLR4 upregulation by DCis essential for the CD8� T-cell response against E. cuniculiinfection.

DISCUSSION

Our results show that TLR4 plays a major role in activationof splenic DC during E. cuniculi infection, which results ininitiation of a protective immune response. While increases inTLR4 transcript and surface protein expression on splenic DCwere observed, the expression of other important TLRs (TLR2and TLR9) remained the same. The data presented in thispaper demonstrate that TLR4 expressed by DC signalsthrough the MyD88 adapter molecule and that in vitro block-ade of this receptor reduces the expression of MHC class IIcostimulatory molecules, as well as IL-12p40 production bythese cells. This significantly decreases the ability of DC toprime an optimal T-cell response against E. cuniculi. Theseresults emphasize the role of the TLR4 molecule in initiatingimmunity against this pathogen.

TLR signaling on DC is involved in the immune responses tonumerous pathogens (18, 36, 39). Specifically, TLR4 has beendemonstrated to be one of the molecules stimulated in severalinfectious diseases (3, 6, 8–10). Additionally, it has been shownthat TLR polymorphism increases susceptibility to certain dis-eases. For instance, Mockenhaupt et al. found that Africanchildren with a TLR4 polymorphism were more susceptible tosevere malaria infections, which showed the importance of thismolecule in initiation of the immune response against P. fal-ciparum (28). Furthermore, it has been reported that a muta-tion in TLR4 increased the risk of invasive meningococcaldisease in infants less than 12 months old (10).

The TLR molecules involved in the priming of the T-cellresponse during E. cuniculi infection have not been well de-scribed. This pathogen has been reported to induce a lessrobust immune response in TLR9�/� mice following oral in-fection (16). The altered immune response in these mutantanimals was attributed to enhancement of the regulatory T-cellresponse by commensal DNA in the intestinal tissues and/or tothe inability of the mice to generate optimal effector T-cellimmunity (16). Recent in vitro studies have demonstrated thatE. cuniculi infection upregulates the TLR2 message in humanmonocyte-derived macrophages (11). However, no increase inthe TLR4 message was observed in this study (11). Although arole for TLR2 in activation of DC during E. cuniculi was notobserved in our study, it is possible that TLR2 may be involvedin activation of other antigen-presenting cells, like macro-phages, as reported in previous studies (3). Alternatively, it isvery likely that TLRs involved in the responses to microspo-ridian infections in human and mouse macrophages or DC aredifferent.

In the present study, we demonstrated that TLR4 signalingis important for generation of robust CD8� T-cell immunityagainst E. cuniculi. Interestingly, TLR4 blockade reducesIL-12 production by E. cuniculi-pulsed DC. A role for IL-12 in

induction of short-lived effector CD8� T cells against intracel-lular parasitic infection has been demonstrated (40). Thus,reduced IL-12 production by DC due to TLR4 blockade maycontribute to the inability of these cells to induce an optimalCD8� T-cell response.

As E. cuniculi and other microsporidia are an ever-increas-ing problem for immunocompromised patients, an effectivevaccination against these pathogens would be advantageous.This study demonstrated that TLR4 is involved in the primingof CD8� T cells, which have been shown to be critical forprotection against E. cuniculi infection (21, 29, 30). The novelfeature of our observations is that they demonstrate the im-portance of TLR4 in elicitation of CD8� T-cell immunityagainst E. cuniculi infection, which has been reported to becritical for protection of hosts against this pathogen. Thesefindings suggest the need to target the TLR4 molecule fordeveloping immunotherapeutic reagents against E. cuniculiand other microsporidian species, such as Enterocytozoon bie-neusi, which cause severe complications in HIV-infected indi-viduals.

ACKNOWLEDGMENTS

We thank Teresa Hawley for her help with flow cytometry.This work was supported by National Institute of Health grant AI

043693 awarded to I.A.K.

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