Lipid Rafts Presentation of Macrophage by Disrupting Affects Antigen Leishmania Donovani

Leishmania donovani Affects Antigen Presentation of Macrophage by Disrupting Lipid Rafts

This information is current as Debabani Chakraborty, Subha Banerjee, Abhik Sen, Kalyan of October 3, 2021. K. Banerjee, Pradip Das and Syamal Roy J Immunol 2005; 175:3214-3224; ; doi: 10.4049/jimmunol.175.5.3214 http://www.jimmunol.org/content/175/5/3214 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2005 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Leishmania donovani Affects Antigen Presentation of Macrophage by Disrupting Lipid Rafts1

Debabani Chakraborty,2* Subha Banerjee,2* Abhik Sen,† Kalyan K. Banerjee,† Pradip Das,† and Syamal Roy3*

Leishmania donovani-infected splenic macrophages and P388D1 (P388D1(I)) failed to activate T cells in response to low dose of exogenous peptide. The membrane fluidity of P388D1(I) was greater than that of the normal counterpart P388D1(N), but could be reduced either by exposing the cell below phase transition point or by loading cholesterol into membrane (L-P388D1(I)), and this was associated with enhanced Ag-presenting ability of P388D1(I). Presentation of endogenous leishmanial Ag, kinetoplastid membrane protein-11, was also defective, but could be corrected by loading cholesterol into membrane. Because membrane rafts are important for Ag presentation at a low peptide dose, raft architecture of P388D1(I) was studied using raft (CD48 and cholera toxin-B) and non-raft (CD71) markers in terms of their colocalization with I-Ad. Binding of anti-CD48 mAb and cholera toxin B Downloaded from subunit decreased significantly in P388D1(I), and consequently, colocalization with I-Ad was not seen, but this could be restored in L-P388D1(I). Conversely, colocalization between I-Ad and CD71 remained unaffected regardless of the presence or the absence of intracellular parasites. P388D1(N) and L-P388D1(I), but not P388D1(I), formed peptide-dependent synapse with T cells quite efficiently and this was found to be corroborated with both intracellular Ca2؉ mobilization in T cells and IL-2 production. This indicated that intracellular parasites disrupt the membrane rafts, possibly by increasing the membrane fluidity, which could be corrected by making the membrane rigid. This may be a strategy that intracellular L. donovani adopts to evade host immune http://www.jimmunol.org/ system. The Journal of Immunology, 2005, 175: 3214–3224.

eishmaniasis is a vector-borne parasitic disease with a show that human blood monocyte-derived DCs could be infected broad range of clinical manifestation from local cutane- with Leishmania donovani (LD) (6), and maturation of mouse L ous lesion to life-threatening visceral disease, mainly bone marrow-derived DCs is inhibited by Leishmania amazonen- caused by differences among Leishmania species and the immu- sis (7). The present study is concerned with the Ag-presenting nological status of the mammalian hosts (1). The parasite exists in ability of M␾ as a general mechanism to understand how intra- two different stages; the flagellated promastigotes are transmitted cellular Leishmania parasites modulate the surface phenomena of with the bite of the sandfly to the mammalian hosts, where they APCs, which, in turn, may affect Ag presentation. by guest on October 3, 2021 4 transform into the amastigote stage. Although macrophage (M␾) The disease visceral leishmaniasis or kala-azar, caused by the are generally considered to be the main or only host cells allowing protozoan parasite LD, is characterized by defective cell-mediated Leishmania to survive and multiply (2), other cells can harbor immunity (8–13), the cause of which is still unknown. Leishma- intact viable parasites at least transiently. For example, Leishmania nia-infected M␾ are unable to present even processing-indepen- major parasites have been observed in polymorphonuclear leuko- dent peptide sequences to T cell hybridoma, and this inability is cytes, fibroblast, and dendritic cells (DC) at different stages of not due to defective MHC expression (14). Recent studies show infection (3). The ability of Langerhans cells and DCs to take up that Leishmania-infected M␾ efficiently stimulate Ag-independent Leishmania is still a matter of debate (4, 5). Murine fetal skin- and Ag-dependent TCR-mediated capping of F-actin in T cells derived DCs are preferentially infected by L. major amastigotes (15). Infected M␾ are less efficient at promoting the sustained T compared with metacyclic promastigotes (5). Recently, studies cell signaling events, but this is not due to altered levels of surface- expressed peptide-MHC complexes (15). *Department of Immunology, Indian Institute of Chemical Biology; and †National Physiological stimulation of T cells by antigenic peptides pre- Institute of Cholera and Enteric Diseases, Scheme XM, Beliaghata, Kolkata, India sented in the context of MHC molecules requires a stable contact Received for publication May 24, 2005. Accepted for publication June 13, 2005. between T cells and APCs to enable the generation of efficient The costs of publication of this article were defrayed in part by the payment of page activation signals (16, 17). The area of contact between the two charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. cells, the immunological synapse, has recently become a fruitful focus of investigation (18, 19). Recent work on the membrane 1 This work was supported by the Council for Scientific and Industrial Research, New Delhi, India (Project CMM002). D.C. and S.B. are recipients of Council of Scientific features that affect presentation of Ags by APCs to T cells indi- and Industrial research fellowships. cates that lipid rafts in the outer leaflet of the membrane lipid 2 D.C. and S.B. contributed equally to the work. bilayer induce clustering of the peptide-MHC complex on the APC 3 Address correspondence and reprint requests to Dr. Syamal Roy, Department of surface. This leads to an effective increase in the surface density of Immunology, 4 Raja S. C. Mullick Road, Kolkata 700032, India. E-mail address: the MHC-Ag and up-regulation of the T cell response (20–22). [email protected] Lipid rafts are cholesterol- or sphingolipid-rich microdomains with 4 Abbreviations used in this paper: M␾, macrophage; LD, Leishmania donovani; CTX-B, cholera toxin B subunit; DC, dendritic cell; DPH, 1,6-diphenyl-1,3,5- high melting temperatures caused by ordered packing of molecules hexatriene; FA, fluorescence anisotropy; HEL, hen egg lysozyme; I-M␾, infected (23). The proposed functions of lipid rafts include cholesterol splenic macrophage; KMP-11, kinetoplastid membrane protein-11; L-P388D1(I), cholesterol-loaded P388D1(I); P388D1(I), infected P388D1 cell; P388D1(N), unin- transport (24, 25), endocytosis (26), signal transduction (27–29), fected P388D1 cell; SLA, soluble leishmanial Ag. potocytosis (30), and pathogen portals (31–34). The concept of

lipid rafts as pathogen portals has gained considerable interest in Mice, infection, and preparation of splenic and peritoneal M␾ recent years. When Plasmodium invades RBC, host cell plasma BALB/c mice were obtained from the animal facility of the institute. membrane lipids are incorporated into the parasite-containing vac- CBA/J mice were a gift from Dr. S. K. Basu (National Institute of Immu- uole, whereas transmembrane proteins are excluded (35). Simi- nology, New Delhi, India). Mice were housed under conventional condi- larly, in Toxoplasma gondii infection, GPI-anchored surface pro- tions, with food and water ad libitum. Animals were used for experimental teins are incorporated into parasitophorous vacuole, whereas host purposes with prior approval of the institutional animal ethics committee. Spleens of BALB/c mice were isolated and macerated between the frosted cell transmembrane proteins are excluded from it (36). Cholesterol ends of a pair of glass slides in PBS (pH 7.2). The cell suspension was then appears to play a key role in organizing the lipid domain, which is layered over Ficoll-Hypaque, and density gradient centrifugation was per- required for the entry of mycobacteria (37) and Leishmania (38) formed at 1600 rpm for 20 min. The cells in the interface were collected into the host cells. Disruption of lipid rafts by sequestration of and washed twice in PBS. The splenic M␾ were purified as previously ␤ described (12). Similarly, peritoneal M␾ of CBA/J mice were isolated as cholesterol after treatment of APCs with methyl -cyclodextrin previously described (53). For infection, 6- to 8-wk-old BALB/c mice were impaired the presentation of peptide-MHC complexes (22). The inoculated with 107 early passage promastigotes via the intracardiac effect could be reversed at high Ag concentrations, indicating that route (51). lipid rafts indeed play an important role in enhancing the T cell Splenic M␾ and P388D1 cell lines were used for in vitro infection 5 response at low ligand concentrations (39). Interestingly, changes studies. Cells (10 ) were allowed to adhere on glass coverslips for3hat 37°C in presence of a 5% CO2 atmosphere, after which the nonadherent in the membrane fluidity of cells occur under a variety of patho- cells were removed by gentle washing with serum-free medium. The ad- logical conditions (40, 41). herent M␾, after overnight incubation in complete medium, were chal- Such changes in fluidity are reflected in reduced T cell-mediated lenged with LD promastigotes at an M␾ to parasite ratio of 1:10 and in- cytotoxicity (42–44) as well as NK cell-mediated target cell lysis cubated further for6hat37°C. Excess parasites were then washed off with serum-free medium. The M␾ were then incubated further for 6, 12, 24, 36, Downloaded from (45). Previously, we have demonstrated that a subset of splenic 48, 60, and 72 h to determine their ability to support intracellular parasite M␾ of Leishmania-infected BALB/c mice display an increase in replication (54). The splenic M␾ and P388D1, infected or not, were used membrane fluidity coupled with defective Ag-presenting function; as APCs to drive T cell hybridomas in the presence or the absence of ␭ this inability to present Ag was not due to the lack of peptide-MHC R12–26 (55). complex formation on the surface of M␾ (11). The present work demonstrates for the first time that during their intracellular life Ag presentation and peptide pulsing cycle, Leishmania parasites disrupt the membrane raft of M␾ by http://www.jimmunol.org/ The APCs were incubated for 24 h with specific peptide and T cell hy- increasing membrane fluidity. This increase in fluidity could be bridoma in complete RPMI 1640 medium in a 37°C incubator. The culture corrected either by exposing the parasitized M␾ to lower than supernatants were analyzed for the presence of IL-2 by growing an IL-2- phase transition temperature or by liposomal delivery of choles- dependent cell line, HT-2, in the supernatants. HT-2 (104/well) was incu- terol, leading to correction in Ag presentation. Thus, Leishmania bated with a 50% concentration of culture supernatant for 24 h. The cells were then pulsed with 1 ␮Ci of [3H]thymidine for the last 18 h (55). The may evade the host immune response by exploiting rafts during incorporation of radioactive thymidine was assessed by a scintillation their intracellular lifecycle. counter (Packard). Uninfected P388D1 (P388D1(N)) and infected P388D1 6 ␮ ␭ (P388D1(I)) cells (10 /ml) were pulsed with 2 M R12–26 peptide for 1 h Materials and Methods at 37°C and extensively washed with serum-free RPMI 1640 medium. by guest on October 3, 2021 Abs and other reagents They were then resuspended in complete RPMI 1640 medium to a cell density of 2 ϫ 105/ml. These peptide-pulsed P388D1(N) and P388D1(I) FITC-conjugated anti-mouse I-Ad, PE-conjugated mAb to mouse CD71 cells were used as APCs to drive peptide-specific T cell hybridomas. Nor- and CD48, biotin-conjugated anti I-Ad, and streptavidin-PE were pur- mal M␾ were pulsed with HEL (1 mg/ml) for 4 h, then washed and kept chased from BD Pharmingen. FBS, the peptide LEDARRLAIYEKK (N- for another 24 h. Finally, cells were stained with FITC-labeled-C4H3, and ␭ ␭ terminal 12–26 aa residues of repressor protein, also defined as R12–26), the extent of peptide-MHC complex present on the cell surface was deter- and RPMI 1640 medium were obtained from Invitrogen Life Technologies. mined by FACS analysis (15). Penicillin-streptomycin, sodium bicarbonate, HEPES, 2-ME, phosphatidylethanolamine, 1,6-diphenyl-1,3,5-hexatriene (DPH), fura 2-AM, paraformaldehyde, and FITC-conjugated cholera toxin B subunit (CTX-B- Measurement of fluorescence anisotropy (FA) FITC) were purchased from Sigma-Aldrich. Cholesterol was obtained from The membrane fluorescence and lipid fluidity of cells were measured fol- Merck. [3H]TdR (sp. act., 6.7 Ci/mmol) was purchased from New England lowing the method described by Shinitzky and Inbar (56). Briefly, the Nuclear. Na 51CrO was obtained from Bhaba Atomic Research Center 2 4 fluorescent probe DPH was dissolved in tetrahydrofuran at 2 mM concen- (sp. act., 65.1Ci/g). Hen egg lysozyme (HEL) was obtained from Roche. tration. To 10 ml of rapidly stirring PBS (pH 7.2), 2 mM DPH solution was Ab C4H3 specific for IAk-HEL (46) was a gift from Dr. R. N. Ger- 46–61 added. For labeling, 106cells were mixed with an equal volume of DPH in main (National Institutes of Health, Bethesda, MD). Ab C4H3 was conju- PBS (C 1 ␮M) and incubated for2hat37°C. Thereafter the cells were gated to FITC as previously described (47). f washed thrice and resuspended in PBS. The DPH probe bound to the mem- Cell lines brane of the cell was excited at 365 nm and the intensity of emission was recorded at 430 nm in a spectrofluorometer. The FA value was calculated ␾ Murine M -like tumor cell P388D1 (48, 49) was used in in vitro experi- using the equation: FA ϭ [(Iʈ Ϫ IЌ)/(Iʈ ϩ 2IЌ)], where Iʈ and IЌ are the ments. T cell hybridoma 9H3.5 (50) of BALB/c origin, specific for fluorescent intensities oriented, respectively, parallel and perpendicular to ␭ R12–26, was a gift from Prof. M. L. Gefter (Massachusetts Institute of the direction of polarization of the excited light (57). Technology, Cambridge, MA). The IL-2-dependent cell line HT-2 was obtained from American Type Culture Collection. All cells were maintained in RPMI 1640 medium supplemented with 10% FCS and 2-ME (5 ϫ Phase transition in relation to Ag presentation 10Ϫ5 M) at 37°C with 5% CO in a humidified atmosphere. 2 In one set, P388D1(N) and P388D1(I) cells were pulsed with 2 ␮M peptide Parasite maintenance and preparation of soluble leishmanial Ag for 1 h, then washed and resuspended in complete RPMI 1640 at a final cell 6 (SLA) density of 10 cells/ml. The cells were incubated at 10, 15, 20, 25, 30, or 37°C for 15 min and then fixed in 1% paraformaldehyde in PBS. They were LD strain AG83 (MHOM/IN/1983/AG83), originally obtained from an In- seeded in a 96-well microtiter plate (Falcon) at a concentration of 2 ϫ 104 dian kala-azar patient (51), was maintained in Golden hamsters as de- cells/well, incubated with 9H3.5 T cell hybridoma (105 cells/well) for 24 h scribed previously (52). Promastigotes obtained after transforming amas- at 37°C, and the IL-2 produced was measured by a previously described tigotes from the spleen of infected animals were maintained in culture in method (55). P388D1(I) cells were pulsed with the peptide at 37°C, then medium 199 (Invitrogen Life Technologies) supplemented with 10% FCS exposed to 15°C for 15 min and fixed with paraformaldehyde (prepulsed). at 22°C. The culture was replenished with fresh medium every 72 h. SLA In another situation, P388D1(I) cells were exposed to 15°C for 15 min, was prepared as described previously (51). then fixed with paraformaldehyde and pulsed with peptide (postpulsed). 3216 L. donovani INFECTION DISRUPTS RAFTS

Liposome preparation and delivery of cholesterol ml. The cell suspension (1 ml) was placed in a continuously stirred cuvette at room temperature in a fluorometer (Hitachi U440). Fluorescence was Liposomes were prepared with cholesterol and phosphatidylethanolamine ␭ ϭ ␭ ϭ ␭ ϭ monitored in real time at ex1 340 nm, ex2 380 nm, and em 510 at a molar ratio of 1.5:1 as previously described (58, 59). Briefly, a thin dry nm at a bandwidth of 10 nm, and the data were presented as the relative film of lipids (5.8 mg cholesterol and 8.0 mg phosphatidylethanolamine) ratio of fluorescence excited at 340 and 380 nm. After ϳ1 min of scanning, was dispersed in 1 ml of RPMI 1640 and sonicated at 4°C three times, 1 5 the peptide-pulsed or unpulsed APC (P388D1(N), P388D1(I), and min each, at maximum output. To alter the fluidity of cells, 10 intact cells L-P388D1(I)) were added to the cuvette, and the ratio of fluorescence was were incubated with liposomes for 12 h at 37°C. The cells with altered monitored (61). fluidity were then washed three times in serum-free RPMI 1640 medium and finally resuspended in 10% FCS containing RPMI 1640. The FA value Statistical variation and presentation of the liposome-treated cells was calculated as previously described (57). Each experiment was performed three to five times, and representative data Analysis of peptide-MHC complex from one set of these experiments are presented; the interassay variation was within 10%. Results are expressed as the mean Ϯ SD of the individual ␾ Peritoneal M of CBA/J mice were infected with LD for 6 h, washed, and set of experiments. kept overnight. In another set, infected M␾ after6hofinfection were treated with cholesterol-rich liposome for overnight. Normal, infected, and cholesterol-loaded infected M␾ were pulsed with HEL (1 mg/ml) for 4 h, Results washed, kept overnight, and stained with C4H3-FITC conjugate for 30 Infection of splenic M␾ and P388D1 cells with LD ␾ ␾ min. In another set, peptide-pulsed infected splenic M (I-M ) were ex- ␾ posed to 15°C for 15 min (prepulse), stained with C4H3-FITC at 15°C for Splenic M were infected with LD, and intracellular parasites 30 min, and fixed with paraformaldehyde in a chilled condition. The extent were enumerated after 6, 12, 24, 36, 48, 60, and 72 h of infection. of binding of C4H3-FITC was analyzed by flow cytometry. After 24 h of infection, 80% of splenic M␾ were found to be ϳ ␾ infected harboring 4 parasites per M (Fig 1). The maximum Downloaded from Confocal microscopy and analysis of conjugate formation number of I-M␾ was observed at 48 h of infection, and at this point P388D1(N), P388D1(I), and cholesterol-loaded P388D1(I) (L-P388D1(I)) the number of parasites per M␾ was six (Fig. 1). P388D1, a mouse cells were harvested, washed, and resuspended in cold wash buffer (PBS/ M␾-like cell, supports Leishmania replication and has been in use ϫ g 0.1% NaN3/1% FBS), centrifuged at 350 for 5 min, and finally resus- ␾ pended in 50 ␮l of wash buffer. Cells were stained with fluorochrome- over the years as a model host instead of primary M culture (49). conjugated Ab according to the manufacturer’s protocol. Briefly, FITC- Enumeration of the number of internalized parasites in P388D1(I) conjugated anti I-Ad was diluted to a predetermined optimal concentration cells after 6, 12, 24, 36, 48, 60, and 72 h of infection showed that 6 http://www.jimmunol.org/ in 50 ␮l of FACS buffer. Cells (10 )in50␮l of wash buffer were added the intracellular parasite count increased up to 48 h. At 24 h, to diluted conjugate, mixed thoroughly by tapping, and incubated at 4°C in Ͼ95% of the cells were infected. The percentage increased slightly the dark. The reaction was stopped by adding 200 ␮l of FACS buffer and washed three times. For staining with second Ab, PE-conjugated anti- at 48 h and then reached a plateau (Fig. 1). At 24 and 48 h, the CD48/CD71 was added as described above. In another set, the above cell average numbers of intracellular parasites were 10–12 and 12–14, types (P388D1(N), P388D1(I), and L-P388D1(I)) were stained with bio- respectively. In the splenic M␾, maximum parasite number was d tinylated anti I-A and streptavidin-PE, then costained with FITC-CTX-B attained at 48 h; in the case of P388D1(I) cells, near-maximal following the protocol described above. For studying the conjugate formation, APCs (P388D1(N), P388D1(I), and L-P388D1(I)), either unpulsed or internalization of parasites was attained at 24 h. After this, studies ␮ ␭ ␾ pulsed with 2 M R12–26 for1hat37°C, were labeled with biotinylated were conducted with 48-h infected splenic M and 24-h infected d anti I-A and then with streptavidin-PE (19). The T cell hybridomas were P388D1 cells as APC. by guest on October 3, 2021 stained with CTX-B-FITC. APC and T cells, mixed at a ratio of 1:5, were resuspended in FACS buffer and allowed to form conjugate for 30 min. The Inability of LD-infected M␾ and P388D1 to present peptide Ag cells were then fixed with 1% paraformaldehyde, mounted with 90% glyc- at a low peptide dose erol on a glass slide, and observed under a laser scanning microscope (LSM 510; Zeiss). Cells found in joint couplets under phase contrast and exhib- The Ag-presenting ability of splenic M␾ infected with LD was iting a zone of colocalization under confocal were considered to have ␭ investigated with increasing concentrations of R12–26. Because formed a synapse. The number of such synapse-forming couplets per 100 our study is concerned with cell surface phenomena associated APCs is presented as percentage of synapse formation. with Ag presentation, we have used the processing-independent ␭ ␾ Anti-kinetoplasid membrane protein-11 (anti-KMP-11) CTL peptide sequence R12–26 to drive T cells (14). I-M failed to response KMP-11-expressing mammalian expression vector construct (pCMV-LIC KMP-11) used for immunization purpose was generated as previously described (60). Nonadherent splenocytes from pCMV-LIC KMP-11 DNA- vaccinated BALB/c mice (3 wk after immunization with two booster doses of 100 ␮g of plasmid, 7 days apart i.m.) stimulated with SLA for 4 wk were used as effectors. The targets were the splenic M␾ (106) of normal and 2-month infected BALB/c mice either treated or not with liposome. These 51 ␮ were labeled with Cr (100 Ci)for1hat37°C in 5% CO2 incubator and washed several times until no gamma irradiation was detected in the supernatant. The effectors and targets were mixed in round-bottomed, 96-well plates (200 ␮l) at various (12:1, 25:1, and 50:1) ratios. After 4-h incubation, 100 ␮l of culture supernatant was collected and counted in triplicate in a liquid scintillation counter (Tri-Carb 2100TR; Packard). Specific lysis was calculated according to the formula: % specific lysis ϭ (sample Ϫ spontaneous release)/(maximum release Ϫ spontaneous release) ϫ 100 (60). Intracellular calcium mobilization in T cells ␾ 2ϩ FIGURE 1. Infection of splenic M and P388D1 cells with LD in vitro. Intracellular Ca mobilization in T cell hybridoma was monitored in re- ␾ sponse to peptide-pulsed and unpulsed APCs. Briefly, T cell hybridomas Splenic M s or P388D1 cells were adhered on coverslips for 3 h and (106/ml) were taken in RPMI 1640 (without FCS), washed once, and re- challenged with LD (cell:parasite ratio, 1:10) for 6 h, followed by incuba- suspended in RPMI 1640 containing 6 ␮M fura 2-AM for 60 min at 37°C tion for varying time periods to establish infection. Coverslips were air- in the dark with gentle shaking. The cells were washed with HBSS and dried, fixed with methanol, and stained with Giemsa, and intracellular par- resuspended in HBSS containing 0.5 ␮M EGTA at a cell number of 106/ asites were enumerated. The Journal of Immunology 3217

activate 9H3.5 at a low peptide concentration, unlike normal splenic M␾. This inability could largely be overcome by loading ␮ ␭ the APC with a high dose of the peptide (10 M R12–26; Fig. 2). Identical observations were made using P388D1(N) and P388D1(I) as APCs (Fig. 3). In some of the subsequent experi- ␭ ments, we pulsed P388D1(N) and P388D1(I) cells with R12–26 peptide. Ag presentation assays using these peptide-pulsed APCs showed results essentially similar to those of assays in which peptide was kept throughout in the culture medium (Fig. 3, inset).

Membrane fluidity under parasitized condition Leishmania-infected M␾ are reported to be unable to present processing-independent peptide to T cell hybridoma (14), and changes in membrane fluidity in effector cells influence T cell-mediated cytotoxicity (43). Because LD-infected splenic M␾ and P388D1 showed identical patterns of response in terms of Ag presentation, the rest of the studies were conducted with P388D1 cells. There- FIGURE 3. Ag-presenting ability of P388D1(N) and P388D1(I) cells to fore, we studied the membrane fluidity of P388D1(N) and T cell hybridoma, 9H3.5. Normal and 24-h parasitized M␾s were tested for P388D1(I) cells in terms of FA using DPH as probe. The observed the ability to activate 9H3.5 T cell hybridoma using increasing doses of ␭R . The readout was the growth of the IL-2-dependent cell line HT-2. Downloaded from decrease in FA (0.35 vs 0.23) due to infection at 24 h (Fig. 4) 12–26 Inset, P388D1(N) and P388D1(I) cells were pulsed with 2 ␮M ␭R indicated an increase in membrane fluidity. 12–26 peptide, and Ag presentation was assayed. Influence of membrane fluidity on Ag presentation If an increase in membrane fluidity in infected cells was indeed observed with 2 ϫ 105 peptide-pulsed P388D1(N) exposed to responsible for defective Ag presentation, the efficiency of Ag pre- 37°C. Interestingly, to obtain an identical response, only 2 ϫ 103 http://www.jimmunol.org/ sentation by normal APCs would be expected to undergo a sharp of 15°C-exposed, peptide-pulsed P388D1(N) were required (Fig. change within a narrow temperature range characteristic of the 5, inset). When the results were expressed as the fold enhancement phase transition of the membrane lipid bilayer (23). P388D1(N) in Ag-presenting ability between the cells exposed to 15 vs 37°C cells were therefore pulsed with 2 ␮M peptide, and aliquots of for a given cell number, this ratio was found to vary between 2.05 cells were exposed to various temperatures, i.e., 10, 15, 20, 25, 30, and 2.8. The transition temperature between the two states was and 37°C for 15 min. These cells were then fixed with 1% para- between 20 and 15°C (Fig. 5). This indicated a clear inverse re- formaldehyde and used as APCs. Corresponding FA values of lationship between membrane fluidity and Ag presentation. Hence- fixed APCs were calculated using DPH as a probe. There was a forth, the cells exposed to a below phase transition temperature

concomitant increase in the FA when cells were exposed to a lower were defined as chilled cells. by guest on October 3, 2021 temperature, indicating a decrease in membrane fluidity (Fig. 5). To study the membrane turnover, if any, that can contribute to Interestingly, Ag presentation was enhanced ϳ2-fold as the incu- defective APC function during infection, P388D1(I) cells were bation temperature was decreased from 20 to 15°C (Fig. 5). The pulsed with peptides either before (prepulse) or after (postpulse) results presented above suggest that there is a significant enhance- fixing with paraformaldehyde. As usual, P388D1(I) failed to in- ment in the potency of APC function when cells are chilled. The duce T cell activation. Interestingly, prepulsing and chilling potency difference may result from better APC function upon chill- P388D1(I) caused a significant enhancement of Ag presentation ing, and this should be reflected in the APC dose-response graph (Fig. 6). An essentially identical response was obtained when post- with a fixed number of T cells. The maximal T cell activation was pulsed and chilled P388D1(I) were used (Fig. 6). When the results

FIGURE 2. Ag presentation by normal and parasitized splenic M␾. FIGURE 4. Measurement of FA of P388D1(N) and P388D1(I) cells. Normal and 48-h parasitized M␾s were tested for their ability to activate T The FA value was measured using DPH as a probe. The ﬂuorophore was cell hybridoma 9H3.5 cells in terms of IL-2 production using increasing excited at 365 nm, emission intensity was recorded at 430 nm, and FA was ␭ doses of R12–26. calculated. 3218 L. donovani INFECTION DISRUPTS RAFTS

FIGURE 5. Effect of phase transition temperature on Ag-presenting ␮ ␭ ability of P388D1(N) cells. Cells were pulsed with 2 M R12–26 for1h FIGURE 7. T cell response to graded doses of peptide-pulsed at 37°C, washed, exposed to various temperatures for 15 min, and then P388D1(N), P388D1(I), and P388D1(I)-15. The Ag-presenting abilities of fixed with 1% paraformaldehyde. With one set of cells, an Ag presentation APCs were assayed after pulsing cells with peptide and fixing them with assay was performed as described in Fig. 2; with the other set, the FA value paraformaldehyde. was calculated. Inset, T cell-stimulating ability of a graded number of peptide-pulsed P388D1(N) exposed to 37°C (N-37) and 15°C (N-15). ered through the liposome to infected cells. This resulted in an increase in the FA value of these cells, i.e., the fluidity decreased Downloaded from compared with cholesterol-undelivered cells, making it compara- were expressed as the fold enhancement in Ag-presenting ability ble to that of normal cells (Fig. 8). The L-P388D1(I) cells showed between the cells exposed to 15 or 37°C for a given cell number, ϳ3-fold enhancement of Ag-presenting ability (Fig. 8). Thus, a this ratio varied between 6.3 and 10 in the case of P388D1(I). decrease in the fluidity of P388D1(I) cells by loading cholesterol Because a decrease in FA due to infection (Fig. 4) is essentially almost corrected the defective Ag-presenting ability. equal to an increase in FA upon chilling (Fig. 5), it was relevant to http://www.jimmunol.org/ know whether chilled infected APC would behave as normal APC Quantification of cell surface peptide-MHC complex at a low peptide dose to drive T cells. In this event, it was observed It has been reported that surface MHC class II molecules are com- that at a low peptide dose, chilled P388D1(I) behaved like parable in normal as well as parasitized M␾ (15). Because Abs that P388D1(N). P388D1(I) that were not chilled failed to stimulate T can detect the ␭-repressor-peptide I-Ad complex are not available, cells, as expected (Fig. 7). we studied the expression of the I-Ak-HEL complex in peri- There are a number of reports stating that fluidity can also be 46–61 toneal M␾ of CBA/J mice using the specific mAb, C4H3 (15, 46). decreased by incorporating cholesterol in the membrane (62–65). Normal and infected M␾ were then pulsed with HEL and fixed Therefore, we were interested in making P388D1 membrane rigid with paraformaldehyde, and the peptide-MHC complex was quan- by liposomal delivery of cholesterol. Because infection of the by guest on October 3, 2021 titated with C4H3-FITC. LD infection did not inhibit cell surface P388D1 cell line leads to an increase in membrane fluidity (Fig. 4), expression of the peptide-MHC complex. Similarly, there was not as seen in the case of cholesterol extraction in other systems (66, much difference in the binding of C4H3-FITC compared with 67), we tried to incorporate cholesterol in the membrane of HEL-pulsed normal M␾. HEL-pulsed infected M␾ exposed to P388D1(I) by liposomal delivery to make the membrane rigid and 15°C or treated with cholesterol-rich liposome showed essentially to study the cell’s Ag-presenting function. Cholesterol was deliv- similar binding with C4H3-FITC as HEL-pulsed normal M␾. This indicated that an essentially identical level of peptide-MHC complex was present on the M␾ surface (Fig. 9).

Presentation of leishmanial Ag We also studied the ability of a cell surface-associated parasite Ag of intracellular origin taken from 2-mo-infected mice as a target of antileishmanial T cells. Because KMP-11 is expressed on the surface of the splenic M␾ of infected mice (data not shown), anti- KMP-11 T cells were generated by priming mice with the KMP-11 DNA construct, and a CTL assay was performed using splenic M␾ from 2-mo-infected mice as targets. There was no lysis when M␾ from normal animal were used as targets (60). Using splenic M␾ from infected mice at an E:T cell ratio of 25:1, there was ϳ16.5% lysis. In contrast, when the splenic M␾ of infected mice were treated with liposomal delivery of cholesterol and then used as a target, there was 36.2% lysis at the above E:T cell ratio. This FIGURE 6. Effect of phase transition temperature on Ag presentation of indicated that Ags derived from intracellular parasites are not pre- P388D1(I). P388D1(I) cells were pulsed with peptide as described in the sented well due to an increase in the fluidity of parasitized M␾ text. The cells were either kept at 37°C (P388D1(I)-37) or exposed to 15°C (Fig. 10). (P388D1(I)-15) for 15 min and then fixed with paraformaldehyde (prepulsing). Similarly, P388D1(I) were incubated at either 37 or 15°C for 15 min, Leishmania infection disrupts lipid rafts, which reappear after fixed with paraformaldehyde, pulsed with 2 ␮M ␭R for1hat37°C, 12–26 cholesterol delivery to infected cells and washed (postpulsing). A graded number of prepulsed and postpulsed APCs was used to drive a fixed number of T cell hybridoma cells, and Because liposomal delivery of cholesterol to P388D1(I) cells de- resulting IL-2 production was assayed. creased membrane fluidity and enhanced Ag-presenting ability, The Journal of Immunology 3219

FIGURE 8. Effect of liposomal delivery of cholesterol on Ag presentation. A, FA values; B, Ag-presenting abilities of P388D1(N), P388D1(I). and L-P388D1(I). The ability of the APCs to drive T cell hybridoma was studied in terms of IL-2 production.

and because cholesterol is known to partition between raft and unique case, we decided to use another raft marker as a probe. The nonraft phases (68, 69), we decided to investigate the raft archi- ubiquitously expressed glycosphingolipid ganglioside, GM1,

tecture under the parasitized condition. For this study, CD48/ which is highly expressed in lipid rafts and CTX-B, binds selec- Downloaded from CTX-B and CD71 were used as raft (70, 71) and non-raft (72) tively with the external components of GM1 (71). Therefore, we markers, respectively. We presumed that disruption of raft under used CTX-B-FITC as a probe to assess the status of rafts in the parasitized condition would prevent colocalization between CD48/ above conditions. Under parasitized conditions, there was a sig- CTX-B and I-Ad, whereas colocalization between CD71 and I-Ad niﬁcant reduction of CTX-B-FITC binding, which could be re- would remain unaffected. We studied the colocalization between stored upon cholesterol loading. In this study we also observed CD48/CTX-B and I-Ad and also between CD71 and I-Ad. From colocalization between I-Ad and CTX-B in the case of http://www.jimmunol.org/ the confocal images, it was apparent that there was no down-reg- L-P388D1(I) cells (Fig. 11B). The above observations support the ulation of I-Ad expression in the above conditions (Fig. 11A). idea that Leishmania infection disrupts raft formation, which can Staining of the three sets of cells (P388D1(N), P388D1(I), and be restored upon liposomal delivery of cholesterol. L -P388D1(I)) with anti-CD48 showed that P388D1(N) and 2ϩ L-P388D1(I), but not P388D1(I), stained well (Fig. 11A). Colo- Formation of APC-T cell conjugates and intracellular Ca calization studies between CD48 and I-Ad showed that this oc- mobilization curred only with P388D1(N) and L-P388D1(I) cells, not with The ability of peptide-unpulsed and -pulsed P388D1(N), P388D1(I) cells (Fig. 11A). P388D1(I), and L-P388D1(I) cells to form synapses with the pep- d

Identical studies were performed in I-A and CD71 to show that tide-specific T cell hybrid was studied. Optimal synapse formation by guest on October 3, 2021 I-Ad could colocalize with nonraft markers. Colocalization be- was observed at ϳ30 min of incubation of APC-T cells. Peptide tween the above markers was observed in all three conditions (Fig. pulsing led to efficient conjugate formation between 9H3.5 and 11A). To confirm that the reduced expression of CD48 is not a P388D1(N)/L-P388D1(I), but not with P388D1(I), and the extent

FIGURE 9. Presence of stable peptide-MHC complex on M␾. Peritoneal M␾ of CBA/J mice were infected with LD for 6 h, washed, and kept overnight (I-M␾). In another set, I-M␾ after6hofinfection were treated with cholesterol-rich liposome overnight (L-M␾-I). Normal M␾ (N-M␾), I-M␾, and L-M␾-I were pulsed with HEL (1 mg/ml) for 4 h, washed, and kept overnight. N-M␾, I-M␾, and L-M␾-I were stained with C4H3-FITC conjugate for 30 min. In another set, peptide-pulsed I-M␾ were exposed to 15°C for 15 min (prepulse), stained with C4H3-FITC at 15°C for 30 min, and fixed with paraformaldehyde in a chilled condition (I-M␾-15). The extent of binding of C4H3-FITC was analyzed by flow cytometry. The intracellular parasite number was five or six per M␾. Green, red, and blue lines indicate isotype control, unpulsed, and HEL-pulsed M␾, respectively. 3220 L. donovani INFECTION DISRUPTS RAFTS

FIGURE 10. In vitro assay of the anti-KMP-11 CTL response. The anti- KMP-11 CTL response was assayed using 51Cr-labeled splenic M␾ from normal BALB/c mice (N), normal M␾ liposome-treated mice (L-N), 2-mo infected mice (I), and liposome-treated M␾ of infected mice (L-I). The Downloaded from results are expressed as the percentage of cell lysis. FIGURE 12. Analysis of conjugate formation and intracellular Ca2ϩ mobilization in T cells. P388D1(N), P388D1(I), and L-P388D1(I) were ␮ ␭ d pulsed with 2 M R12–26 and stained with biotinylated anti I-A , followed of conjugate formation was 75, 77, and 15%, respectively. Thus, by streptavidin-PE. These APCs were mixed with CTX-B-FITC-stained synapse formation is severely compromised with P388D1(I), and 9H3.5 and kept at 37°C for 30 min, and conjugate formation was studied this can be corrected by loading cholesterol. In the absence of the by both phase contrast and confocal microscopy. The results were also

peptide, a basal level of conjugate formation was observed (ϳ2%). expressed as the percentage of synapse formation. To study intracellular http://www.jimmunol.org/ 2ϩ The picture was somewhat similar when intracellular Ca2ϩ mobi- Ca mobilization, T cells (9H3.5) were loaded with fura 2-AM and then mixed with the above APCs. Synapse formation and induction of intracel- lization in the T cell was studied in response to peptide-pulsed and 2ϩ 2ϩ lular Ca mobilization by P388D1(N) (A), P388D1(I) (B), and -unpulsed APCs. Intracellular Ca mobilization was observed ϩ L-P388D1(I) (C) are shown. No Ca2 mobilization and Ͻ2% synapse for- when T cells were stimulated with peptide-pulsed P388D1(N) and mation were observed with unpulsed APCs. The arrow indicates the addition of APCs to the fura 2-AM-loaded T cells.

L-P388D1(I). Interestingly, only a slight mobilization occurred when stimulation was performed with P388D1(I) (Fig. 12). There by guest on October 3, 2021 was no mobilization when T cells were stimulated with unpulsed APCs.

Discussion Our study clearly showed that I-M␾ and P388D1(I) decreased the magnitude of T cell activation at a low dose of peptide, but both responses were essentially identical with that of their normal counterpart at a high peptide dose. Our study was concerned with cell surface phenomena of Ag presentation; therefore, we used a pro- ␭ cessing-independent peptide sequence ( R12–26) to activate T cells (14). Because parasitized M␾ are capable of activating T cells only at a high peptide dose, it is possible that during the active stage of the disease, when there is a high level of circulating parasite Ag, there may not be any defect in Ag presentation. Identification of free leishmanial Ag(s) in the circulation was not possible on many occasions even in confirmed kala-azar patients (73). Similarly, de- tection of free uncomplexed Ag in the circulation of cotton rats infected with LD has not been successful (74). In many systems the presence of specific Ab impedes the de- tection of free Ag in serum due to the formation of immune complexes (74). Previously we demonstrated that a subset of splenic M␾ of LD-infected BALB/c mice showed increased membrane fluidity and defective Ag-presenting ability (11). The question arises of whether the rigidity of the membrane lipid bilayer is a FIGURE 11. Appearance of lipid rafts after cholesterol delivery in critical determinant of the Ag-presenting ability of M␾ in vitro. P388D1(I) cells. A, P388D1(N), P388D1(I), and L-P388D1(I) cells were stained with anti-I-Ad-FITC and anti-CD48-PE or anti-CD71-PE. B, The increase in membrane fluidity in P388D1(I) was indicated by P388D1(N), P388D1(I), and L-P388D1(I) were stained with CTX-B-FITC a significant decrease in FA. However, the inverse correlation be- and costained with biotinylated anti I-Ad, followed by streptavidin-PE. tween membrane fluidity and Ag presentation might be a coinci- Cells were fixed in 1% paraformaldehyde and observed under a confocal dence without mechanistic significance. To explore this possibil- laser scanning microscope. Yellow fluorescence depicts colocalization. ity, we examined the effect of a physical perturbation, such as The Journal of Immunology 3221

temperature, on the Ag-presenting ability of P388D cells. Such an cholesterol loading. It may be recalled in this connection that class effect is likely to be more amenable to interpretation than that I MHC molecules become raft associated under the influence of caused by infection. For this purpose, the normal and infected cells some pathogens (32). Because loading of cholesterol in the were exposed to a temperature of 37°C, the physiological temper- infected M␾ enhances Ag presentation, it appears that infection ature at which the bulk of the lipid bilayer is in a fluid, liquid leads to an increase in membrane fluidity, and this can be crystalline state, allowing lateral mobility of membrane compo- corrected by making the membrane rigid. The disruption of nents, and to 15°C, a temperature at which the bilayer, especially these microdomains or rafts by methyl-␤-cyclodextrin treat- the cholesterol-sphingolipid domain rich in saturated acyl chains, ment, which specifically extracts cholesterol, at a limiting exists in an ordered, solid, gel-like state, causing severe restriction concentration of Ag dramatically inhibits Ag presentation, in lateral mobility (75). With P388D1(N), we observed a which can be overcome by loading the APCs with exceptionally significant decrease in membrane fluidity at 15°C, as indicated high doses of Ag (22). Thus, raft concentrates MHC class II by an increase in FA, coupled with a 2-fold increase in Ag- molecules into microdomains, which allows efficient Ag pre- presenting ability. The effect of incubation at low temperature sentation at low ligand densities (22). was even more marked with P388D1(I); the enhancement in The inability of P388D1(I) to present Ag at low ligand densities Ag-presenting ability was 6.3- to 10-fold. The mechanism of may be due to disruption of rafts brought about by intracellular LD enhanced Ag presentation by chilled APCs is not clear. There is parasites. Because liposomal delivery of cholesterol resulted in a a report that conjugate formation between T cells (influenza decrease in membrane fluidity and enhancement of the Ag-pre- virus hemagglutinin peptide specific) and APC (A20 B 317–329 senting ability of P388D1(I) cells, we hypothesize that intracellular cells) is marginally increased by exposing APCs to 5°C (76). Thus, a decrease in FA is essentially equal to its increase upon parasites lead to the disruption of rafts by an as yet unidentified Downloaded from chilling, and the T cell responses of P388D1(N) and chilled mechanism that can be reversed upon cholesterol loading. Loading ␾ P388D1(I) at a low peptide dose were also identical. This of cholesterol in infected M showed similar binding of C4H3- ␾ association lends credence to the idea that an increase in FITC, which was comparable in infected M , as in infected and ␾ membrane fluidity may be one of the important underlying chilled M . This observation indicated that there was no overall mechanisms of the defective T cell response in leishmaniasis. change in the quantum of peptide-MHC complex. One of the ex- The inability to present peptide was not due to membrane turn- planations for the increased potency produced by either chilling or http://www.jimmunol.org/ over, because an identical response was obtained if P388D1(I) was loading cholesterol is a change in the interaction between the pep- fixed either before pulsing with peptide (postpulsing) or after (pre- tide-MHC complex and TCR. Cholesterol is thought to serve as a pulsing). To determine whether a peptide-MHC complex is indeed spacer between the hydrocarbon chains of the sphingolipid and to present on the cell surface, we analyzed this complex. Because function as a dynamic glue that maintains the raft assembly (68). d ␭ there is no specific Ab available to quantitate the I-A - R12–26 In addition, cholesterol promotes the coexistence of solid, gel-like k complex, we used C4H3, an Ab specific for I-A -HEL46–61 (46). as well as fluid, liquid crystalline domains of the membrane and Bindings of C4H3 to HEL-pulsed I-M␾ and chilled I-M␾ were thereby prevents collapse of the membrane into either gel or fluid comparable to its binding to normal M␾. This observation rein- phases with a change in temperature (78). In other words, choles- by guest on October 3, 2021 forced the above finding that a stable peptide-MHC complex was terol moderates the effect of temperature shock. We may recall in formed when M␾ were infected. A similar observation has been this context that the lowering of temperature caused a significant reported by another group (15). Because loading of cholesterol is increase in the Ag-presenting ability of infected cells compared more physiological to modulate the fluidity of biological mem- with normal cells. brane (68) and steady state FA in model membrane is a sensitive Thus, we studied the raft architecture in P388D1(N), P388D1(I), measure of cholesterol dependent ordering (77), we studied the and L-P388D1(I). It is known that MHC class II molecules are effect of cholesterol loading on Ag presentation. The lipid order both raft and nonraft associated (20). Using CD48 and CTX-B as detected by DPH-PC anisotropy in plasma membrane vesicle is raft markers (70, 71) and a transferrin receptor (CD71) as a nonraft highly sensitive to the amount of cholesterol, further strengthening marker (72), we studied the colocalization of I-Ad molecules. Cu- its utility as a quantitative indicator of lipid raft content within riously, both CD48-PE- and CTX-B-FITC stained P388D1(N) and fluid membranes (77). We therefore made use of liposomal deliv- L-P388D1(I) cells effectively, but not P388D1(I) cells. Interest- ery of cholesterol to make the membrane rigid. There is a report ingly, I-Ad molecules colocalized with CD71, but not with CD48/ that increase in cholesterol in the extracellular milieu may augment CTX-B, in P388D1(I), suggesting the disruption of rafts due to the Ag presentation by modulating the expression of HLA-D region presence of intracellular LD. The raft became evident again from products on APCs (69). Loading of cholesterol in P388D1(I) in- the colocalization between I-Ad and CD48/CTX-B, when deed decreased the membrane fluidity with concomitant increase P388D1(I) cells were loaded with cholesterol. This suggests that in Ag presenting ability. We then asked whether the results observed with exogenously the presence of intracellular parasites somehow disrupts the raft added Ag were true with endogenously derived parasite Ag of structure, which can be restored upon cholesterol loading. Natu- infected M␾ and studied the presentation of KMP-11 to anti- rally the question arises of which physical parameters of a biolog- KMP-11 CTLs (60). KMP-11, a surface membrane protein ex- ical membrane are influenced by cholesterol? Membrane choles- pressed in both amastigote and promastigote forms of the parasite, terol regulates membrane-embedded receptors in terms of their is emerging as a potential vaccine candidate (60). Results from our affinity state, the binding capacity and signal transduction (79). study clearly demonstrated that splenic M␾ from infected BALB/c Most important, cholesterol may stabilize receptors in defined con- mice were specifically recognized and lysed by anti-KMP-11 T formations related to their biological functions (79). Cellular cho- cells only when they were treated with cholesterol in vitro. This lesterol levels affect the shedding of IL-6R (80) and CD30 (81), indicated that despite the presence of KMP-11 in infected M␾,as which may be mediated by increased accessibility of the protease studied by immunofluorescence using anti-KMP-11 Ab (data not to the receptor cleavage site, and depletion of cholesterol from the shown), Ag presentation was rendered defective by altered mem- plasma membrane reduces the amount of MHC class II transferred brane fluidity in pathological conditions, which can be reversed by onto the T cells (82). 3222 L. donovani INFECTION DISRUPTS RAFTS

A very recent study related to the effect of varying cholesterol Acknowledgments k concentrations on GPI-linked and native I-E showed that the dif- We thank Drs. Damo Xu and Farrokh Modabber for critically reviewing fusion coefficients of both are dependent on cholesterol concen- the paper. tration. At a low cholesterol concentration, the diffusion coefficients are reduced up to a factor of 60 for native and 190 for GPI-linked I-Ek. The effect is reversed after cholesterol reintro- Disclosures duction (83). Although the lateral mobility of cell surface macro- The authors have no financial conflict of interest. molecules under the parasitized condition has not been investi- ␾ gated to date, increased ruffling activity has been observed in M References at the site of phagosome formation during Leishmania parasite 1. Parson, R. D., D. A. Wheeler, L. H. Harrison, and H. D. Kay. 1983. The immu- uptake (84). There is a report that Abs specific for a distinct CCR5 nobiology of leishmaniasis. Rev. Infect. Dis. 5: 907–927. epitope lost their ability to bind to the cell surface after cholesterol 2. Alexander, J., and D. G. Russell. 1992. The interaction of Leishmania species Adv. Parasitol. extraction to varying degrees (85). The cause of the reduced ability with macrophages. 31: 175–254. 3. Beil, W. J., G. Meinardus-Hager, D. C. Neugebauer, and C. Sorg. 1992. Differ- of anti-CD48 and CTX-B to bind to P388D1(I) is not known, but ences in the onset of the inflammatory response to cutaneous leishmaniasis in it is tempting to speculate that there is a decrease in either their resistant and susceptible mice. J. Leukocyte Biol. 52: 135–142. number or their accessibility or, perhaps, a combination of both. 4. Konecny, P., A. J. Stagg, H. Jebbari, N. English, R. N. Davidson, and S. C. Knight. 1999. Murine dendritic cells internalize Leishmania major promas- Previously, we have demonstrated that splenic M␾ of late stage- tigotes, produce IL-12 p40 and stimulate primary T-cell proliferation in vitro. infected mice showed altered morphology, as evident from scan- Eur. J. Immunol. 29: 1803–1811. 5. von Stebut, E., Y. Belkaid, T. Jakob, D. L. Sacks, and M. C. Udey. 1998. Uptake ning electron microscopic studies (11). The cells were reported to of Leishmania major amastigotes results in activation and interleukin 1 release Downloaded from undergo a change in shape after cholesterol depletion (83). from murine skin-derived dendritic cells: implications for the initiation of anti- For both the initiation and the termination of the cognate im- leishmanial immunity. J. Exp. Med. 188: 1547–1552. 6. Amprey, J. L., G. F. Spa¨th, and S. A. Porcelli. 2004. Inhibition of CD1 expression mune response, the formation of an immunological synapse be- in human dendritic cells during intracellular infection with Leishmania donovani. tween T cells and APC is recognized as a key event (18, 19). The Infect. Immun. 72: 589–592. mature synapse lasts for several hours and is thought to be impor- 7. Prina, E., S. Z. Abdi, M. Lebastard, E. Perret, N. Winter, and J. C. Antoine. 2004. Dendritic cells as host cells for the promastigote and amastigote stages of Leish-

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