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The Journal of Immunology, 2003, 171: 6706-6713.
Copyright © 2003 by The American Association of Immunologists

Leishmania-Induced Inhibition of Macrophage Antigen Presentation Analyzed at the Single-Cell Level 1

Courtney L. Meier, Mattias Svensson and Paul M. Kaye2

Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, United Kingdom


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
A number of studies have previously examined the capacity of intracellular Leishmania parasites to modulate the capacity of macrophages to process and present Ags to MHC class II-restricted CD4+ T cells. However, the bulk culture approaches used for assessing T cell activation make interpretation of some of these studies difficult. To gain a more precise understanding of the interaction between Leishmania-infected macrophages and effector T cells, we have analyzed various parameters of T cell activation in individual macrophage-T cell conjugates. Leishmania-infected macrophages efficiently stimulate Ag-independent as well as Ag-dependent, TCR-mediated capping of cortical F-actin in DO.11 T cells. However, infected macrophages are less efficient at promoting the sustained TCR signaling necessary for reorientation of the T cell microtubule organizing center and for IFN-{gamma} production. A reduced ability to activate these T cell responses was not due to altered levels of surface-expressed MHC class II-peptide complexes. This study represents the first direct single-cell analysis of the impact of intracellular infection on the interaction of macrophages with T cells and serves to emphasize the subtle influence Leishmania has on APC function.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Macrophages have many roles in the immune system, including acting as APCs and as effector cells in antimicrobial immunity. Nevertheless, many important pathogens of humans, including those that cause tuberculosis, leishmaniasis, toxoplasmosis, and Chagas’ disease, all seek residence in macrophages. Such a lifestyle necessitates the development of strategies to avoid immune attack (1, 2, 3, 4). Although dendritic cells are involved in the initial priming of the anti-leishmanial immune response (5) and may sustain short-term survival of Leishmania parasites, macrophages are the major site for parasite replication. Consequently, the ability of macrophages to process and present Leishmania Ags is thought to be necessary for their efficient interaction with effector T cells and the focused delivery of cytokines which induce leishmanicidal activity (3, 6, 7, 8, 9). A number of studies have, therefore, investigated the Ag-presenting potential of Leishmania-infected macrophages, although often with conflicting results (4, 8, 10, 11, 12, 13, 14, 15, 16). The interpretation of most of these studies is also difficult. T cell function has usually been measured after 24–96 h of coculture, a time frame over which the production of regulatory cytokines and other mediators by Leishmania-infected macrophages (1, 8, 17) may have profound effects on T cell response. Furthermore, the heterogeneous infection levels obtained in vitro make it impossible to determine with confidence whether T cells are interacting with infected or uninfected macrophages in these bulk culture experiments. To overcome these difficulties requires an analysis of Ag presentation at the single-cell level.

T cell activation following contact with Ag-bearing APC is now recognized to be a complex and highly ordered event, involving both Ag-independent adhesion events (18, 19, 20) and Ag-dependent TCR-driven events (20, 21, 22, 23). Current models of T cell activation suggest at least two sequential stages occur. The first involves small numbers of TCR molecules signaling through TCR-associated proteins such as Vav and Fyn (24). These events potentiate integrin-based adhesion and actin-dependent accumulation of lipid rafts (24, 25), a prerequisite for the formation of a three-dimensional supramolecular activation complex at the APC-T cell contact site (often referred to as the immunological synapse) (22, 25, 26, 27). This structure facilitates extended serial TCR engagement, the further recruitment and spatial arrangement of downstream signaling molecules, and completion of a three-dimensional, actin-based cytoskeletal platform (27, 28, 29, 30). Reorientation of the T cell microtubule-organizing center (MTOC)3 also occurs and is a rapid downstream event that is strictly dependent upon TCR signaling (20). MTOC reorientation is believed to be important for focusing cytokine release at the target cell surface (31, 32). Although generally accepted to be central to the processes of T cell activation, most data on the immunological synapse have been obtained using transfected fibroblasts, transformed B cells, and immobilized MHC-peptide complexes.

In this report, we describe the results of studies of Ag presentation by control and Leishmania-infected macrophages performed at the single-cell level using cytoskeletal rearrangements as well as IFN-{gamma} production to monitor T cell activation. We show that infection of macrophages significantly inhibits their capacity to provide optimal TCR signaling to Ag-specific effector T cells. Our data also suggest a novel mechanism by which other intracellular pathogens, in addition to Leishmania, may influence host APC function.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice and parasites

BALB/c (H2d; Tuck and Company, Battlesbridge, U.K.) and H2-congenic BALB.K (H2k; Harlan Breeders, Bicester, U.K.) mice were housed under conventional conditions with food and water ad libitum. Breeding pairs of DO.11-SCID mice were kindly provided by Dr. F. Powrie (Nuffield Department of Surgery, Oxford, U.K.), and bred and maintained under barrier conditions at the London School of Hygiene and Tropical Medicine. Leishmania donovani (LV9) was maintained by serial passage in Syrian hamsters. Amastigotes were isolated from infected spleens, as previously described (33), and used to infect macrophage cultures within 48 h of isolation. All animal care and experimental procedures were in accord with U.K. Home Office requirements.

Isolation and generation of T cells and macrophages

DO.11-transgenic T cells, specific for OVA323–339 in the context of H2Ad, were obtained from the spleens of DO.11-SCID mice by passage through a nylon mesh sieve. After washing, cells were treated with Gey’s solution to lyse erythrocytes and resuspended in complete RPMI 1640 medium (RPMI with 50 µM 2-ME, 2 mM L-glutamine, 1 mM sodium pyruvate, 100 U/ml penicillin, and 100 µg/ml streptomycin; all from Life Technologies, Paisley, U.K.) supplemented with 10% heat-inactivated FCS, unless otherwise stated. Macrophages and dendritic cells were removed by adherence for 2 h at 37°C. DO.11 T cells were purified using anti-CD4 magnetic beads (Miltenyi Biotech, Surrey, U.K.) and confirmed as monoclonal using anticlonotype mAb KJ1-26 (34). Activated effector DO.11 T cells were generated by culture with 400 ng/ml OVA323–339 in the presence of 3000-rad irradiated BALB/c splenocytes for 7 days, followed by OVA323–339 and 50 U/ml recombinant human IL-2 (supplied by the National Institutes of Health AIDS Research and Reference Reagent Program) for 5 days. After two rounds of stimulation, cells were expanded in rIL-2 for another 9 days and then frozen in LN2 until use. Thawed DO.11 cells were washed, resuspended in complete RPMI 1640, and added directly to assays. Murine bone marrow-derived macrophages (BMM{phi}) were obtained by culturing bone marrow cells from BALB/c or BALB.K mice for 6 days in complete DMEM supplemented with M-CSF (10% L cell conditioned medium) and 20% heat-inactivated FCS. M-CSF was removed from the culture medium after day 6, and adherent BMM{phi} were then grown on 13-mm glass coverslips (5 x 104/coverslip) in 24-well plates. For infection experiments, L. donovani or heat-killed L. donovani (65°C for 15 min) were added at a multiplicity of infection of 10:1 or 15:1 (1 h at 37°C). Uninfected and infected BMM{phi} were then cultured in M-CSF-free culture medium containing 50 U/ml rIFN-{gamma} for another 48 h. This dose of IFN-{gamma} induces class II expression (verified in each experiment by staining with anti-H2Ad,bEd,k,r mAb M5/114), but not leishmanicidal activity (Ref. 35 and data not shown).

Formation of APC-T cell conjugates

Eighteen hours before T cell conjugation, BMM{phi} were pulsed with 5 µg/ml OVA323–339 (synthesized by Genosphere Biotechnologies, Paris, France) or the indicated concentration of native OVA (Sigma-Aldrich, Poole, U.K.). For blocking studies, Ag-pulsed BMM{phi} were incubated before conjugation with 50 µg/ml mAb MK-D6 (mouse IgG2a anti-H2Ad; American Type Culture Collection, Manassas, VA) or 14.4.4S (mouse IgG2a anti-H2Ed,k,p,r; American Type Culture Collection), or with 33 µg/ml CTLA4-Ig (gift from Dr. P. Lane, University of Birmingham, U.K.) or control human IgG1 (Sigma-Aldrich) for 45 min at 37°C. For raft disruption, BMM{phi} were washed twice with HBSS, then incubated with 60 µg/ml nystatin in HBSS for 30 min at 37°C. BMM{phi} were then fixed with 1% paraformaldehyde for 15 min in the presence of nystatin to prevent the rapid reversibility of the drug’s effect on membrane rafts. Cells were then washed four times with HBSS, quenched for 15 min with 100 mM L-lysine in HBSS to remove unreacted aldehydes, washed twice with HBSS, and used in conjugate formation. To form T cell-BMM{phi} conjugates, BMM{phi} adherent to 13-mm coverslips contained in 24-well plates were mixed at a 1:1 ratio with effector T cells, centrifuged at 250 x g for 5 min, and then incubated for 30 min at 37°C. Coverslips were then washed twice with warm PBS or HBSS, fixed with 4% paraformaldehyde for 60 min, and processed for immunofluorescence.

Analysis of T cell responses at the single-cell level

T cell activation was monitored in individual T cells bound to BMM{phi}, using immunofluorescence microscopy. Paraformaldehyde-fixed conjugates were quenched with 50 mM NH4Cl in PBS, and then blocked and permeablized by incubation with 1.5% v/v normal goat serum in 0.1% v/v saponin in PBS. Cells were stained with rat anti-yeast {alpha} tubulin (Serotec, Oxford, U.K.) followed by FITC-conjugated goat anti-rat IgG (ICN Pharmaceuticals, Basingstoke, U.K.) and/or with BODIPY-650-conjugated phalloidin to reveal filamentous actin (F-actin) (Molecular Probes, Eugene, OR). Coverslips were mounted in Vectashield + 4',6'-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Burlingame, CA) or 50% v/v glycerol in PBS, and visualized with a x63 (NA 1.4) Plan-Apochromat oil immersion objective using a Zeiss Axioplan LSM 510 confocal microscope (Oberkochen, Germany). Images shown are single optical slices (0.8–1.0 µm) or projections of three optical slices. F-actin polarization was scored as a bright band of fluorescence at the cell-cell contact zone. The T cell MTOC was considered reoriented if it was located within the third of the cell proximal to the cell-cell contact site, resulting in a background in this assay of 33% due to chance MTOC alignment (20). Bound T cells 100–150(100–150) were scored per experimental group off multiple coverslips and p values were calculated using an unpaired Student’s t test or {chi}2, as appropriate. For analysis of infected BMM{phi}-T cell conjugates, infected BMM{phi} were identified by direct visualization under Nomarsky imaging or following staining of nuclei with DAPI or propidium iodide.

Intracellular IFN-{gamma} was determined in conjugated T cells by incubation of preformed conjugates for 30 min as above, followed by a further 2-h incubation at 37°C in 10 µg/ml brefeldin A. Cells were then washed, permeabilized, and blocked as described (33). Primary Ab for detection of IFN-{gamma} was a polyclonal rabbit Ab, made by hyperimmunization with recombinant murine IFN{gamma} (36). Ab binding was detected using Alexa 546-conjugated, highly cross-adsorbed goat anti-rabbit IgG (H + L) (Molecular Probes). Cells were counterstained with 1.5 µg/ml DAPI, FluoGrade (Molecular Probes), and mounted in 50% v/v glycerol in PBS. Minimal background staining was observed using control rabbit IgG, and no specific staining was observed in the absence of Ag.

Evaluation of MHC expression and Ag processing capacity of BMM{phi}

L. donovani amastigotes were labeled for 30 min with 10 µM CellTracker Green CMFDA (Molecular Probes) before infection. This treatment does not affect infectivity or survival of amastigotes in BMM{phi} (data not shown). BALB.K derived BMM{phi} (H2Ak) were then infected at a multiplicity of infection of 10:1 in suspension for 1 h at 37°C. Infected BMM{phi} were washed to remove excess amastigotes, and cultured at 1 x 106/ml in M-CSF free culture medium containing 50–100 U/ml rIFN{gamma} for 48 h. Cells were pulsed with 1 mg/ml hen egg lysozyme (HEL; Fluka, Buchs, Switzerland) for 18 h before FACS staining. Ag-pulsed and control BMM{phi} were surface-stained with biotinylated C4H3 (specific for H2Ak/HEL46–61) (37), biotinylated mouse anti-H2Ak (10–3.6.2), or a biotinylated isotype control. Primary mAbs were detected with streptavidin-PE (Sigma-Aldrich). Samples were analyzed with a FACScan cytometer and CellQuest software (BD Biosciences, Mountain View, CA) and uninfected and infected macrophages discriminated by gating using CMFDA.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Leishmania infection does not compromise the ability of macrophages to induce F-actin reorganization in T cells

First, we established a model system in which we could use a BMM{phi} as APC and visualize its interaction with effector T cells at the single-cell level. DO.11 TCR transgenic T cell blasts (specific for OVA323–339 in the context of H2Ad) bound avidly to bone marrow-derived BMM{phi} obtained from syngeneic BALB/c mice, even in the absence of cognate Ag. As shown in Fig. 1, A and B, ~60% of conjugates showed F-actin polarization at the macrophage-T cell contact site. Visually, this was predominantly composed of T cell-associated F-actin, an interpretation supported by the lack of F-actin accumulation at the site of contact between neighboring BMM{phi} (Fig. 1B). When BMM{phi} were pulsed overnight with OVA and then allowed to form conjugates with DO.11 T cells, the frequency of T cells demonstrating F-actin polarization increased significantly to almost 80% (Fig. 1A). This assay faithfully reproduces the features of Ag-independent and Ag-dependent F-action polarization described by others using B cells or transfected fibroblasts as APC (20, 23). We then proceeded to determine the impact of L. donovani infection on BMM{phi} function. BMM{phi} were infected for 48 h with L. donovani, a period of time which allowed for the establishment of a stable infection (1–5 amastigotes/cell) and degradation and clearance of any dead parasites from the initial challenge. When DO.11 T cell blasts were added to these infected macrophages, we found no significant difference in the overall number of T cell-BMM{phi} conjugates formed (data not shown). More importantly, when T cells formed conjugates with infected BMM{phi}, they received identical signals for both Ag-independent and Ag-dependent polarization of F-actin (Fig. 1A). These data indicated that infection with L. donovani neither disrupted the adhesive properties of macrophages nor inhibited their ability to induce TCR-mediated enhancement of F-actin polarization in T cells.



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  		FIGURE 1. L. donovani infection of BMM{phi} selectively inhibits subsequent T cell activation. Control and L. donovani-infected BMM{phi} were pulsed with OVA ({blacksquare}) or left in medium alone ({square}) for 18 h and then allowed to form conjugates with activated DO.11 T cells. A, Bound T cells were scored for the presence of a bright band of polarized actin at the cell-cell contact site. B, Representative confocal image of a three cell conjugate consisting of two macrophages and one DO.11 T cell (lower right cell), showing F-actin polarization (false-colored green). Host and parasite nuclei are counterstained with propidium iodide (red). C, Bound T cells were scored for whether the MTOC was found in the proximal third relative to the contact site, giving a background of 33% resulting from chance orientation. D, Representative example of negative MTOC response on L. donovani-infected BMM{phi}: rat anti-{alpha}-tubulin, green; BODIPY 650 phalloidin, false-colored red. A single amastigote is identified in the corresponding Nomarsky image (arrowhead). Scale bar equals 10 µm. Data in A and C were obtained from scoring at least 150 T cells per group and are representative of at least five independent experiments. Statistical analysis was performed using the Student t test. Values of p between individual groups are shown.

 
Leishmania infection inhibits the capacity of BMM{phi} to induce TCR-mediated MTOC reorientation

Changes in MTOC position are known to occur during T cell activation and are indicative of the site of TCR engagement (20, 38). Therefore, we evaluated MTOC reorientation in bound T cells by staining with Abs specific for {alpha}-tubulin. The scoring system used introduces a theoretical background level of response (due to chance orientation) of 33%. Taking this into account, we observed no Ag-independent reorientation of the MTOC in T cells bound to BMM{phi} (Fig. 1C). These data are in keeping with previous reports that MTOC reorientation is directly linked to sustained TCR signaling and does not occur following adhesive interactions (20, 38). In the presence of Ag, >60% of T cells had reoriented their MTOC toward the contact site with uninfected BMM{phi}. It is noteworthy that, as in B cell-T cell conjugates (39), the MTOC in BMM{phi} did not reorient to the T cell contact site (data not shown). Strikingly, in five independent experiments in which we subsequently evaluated T cell activation on Leishmania-infected BMM{phi}, we found a significant reduction in MTOC reorientation (Fig. 1, C–D). Thus, within 30 min of conjugate formation, T cells can discriminate between infected and uninfected targets. Whereas T cells respond to infected BMM{phi} with normal F-actin polarization, the frequency of T cells able to reorient their MTOC is significantly reduced. These data are the first to directly demonstrate at the single-cell level that L. donovani-infected BMM{phi} are selectively deficient in their ability to trigger early events in T cell activation. To determine whether parasite viability was a prerequisite for such inhibition to occur, we also pulsed BMM{phi} with heat-killed L. donovani. As dead amastigotes are rapidly degraded in the phagosome and thus not identifiable 24–48 h later (data not shown), we used a high multiplicity of infection (10:1) to try to ensure that almost all BMM{phi} were initially infected. In this experiment, 78 ± 1% of uninfected BMM{phi} rearranged their MTOC in the presence of OVA (compared with 37 ± 3% in the absence of Ag), and this was reduced to 43 ± 3% following infection with live parasites (p = NS vs no Ag; p < 0.001 vs uninfected plus Ag) and 54 ± 5% following pulsing with killed amastigotes (p < 0.01 vs no Ag; p < 0.03 vs live infection plus Ag). Hence, parasite-derived products released during degradation of dead amastigotes may also inhibit the MTOC response of bound T cells.

MTOC reorientation requires a higher threshold of TCR stimulation than F-actin polarization

Given this striking effect of Leishmania infection, we proceeded to further characterize the requirements for MTOC reorientation following BMM{phi}-T cell conjugate formation. First, we confirmed that MTOC reorientation required TCR occupancy. Addition of specific anti-H2Ad, but not control anti-H2Ed mAb, completely inhibited MTOC reorientation under these conditions (Fig. 2A). Second, as we have previously demonstrated that L. donovani infection can inhibit the expression of CD80 expression on macrophages (40), we determined whether the MTOC response was dependent upon CD28-mediated costimulation. As shown in Fig. 2B, the addition of CTLA4-Ig to BMM{phi}-T cell cocultures had no effect on the frequency of responding T cells. Thus, CD28 signaling is not required for inducing MTOC reorientation under these conditions, probably reflecting the lower activation threshold and relative costimulation independence of effector T cells (3). Finally, it has recently been reported that Leishmania-infected macrophages display altered membrane fluidity, with changes in the distribution and activity of membranous projections (41). However, macrophage membrane mobility also did not appear to be necessary for activating T cells for a MTOC response, as prefixation of macrophages with paraformaldehyde had no effect on this event (Fig. 2C).



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  		FIGURE 2. Characterization of the T cell cytoskeletal responses elicited by BMM{phi}. BMM{phi} were pulsed for 18 h with 100 µg/ml OVA ({blacksquare}) or left untreated ({square}) and T cell MTOC reorientation was subsequently determined. BMM{phi} were preincubated with (A) anti-H2Ad or control anti-H2Ed (B) CTLA4-Ig or control human IgG1, or (C) were prefixed with 1% paraformaldehyde. D, BMM{phi} were pulsed with various concentrations of OVA and after conjugation, T cells were scored for either actin polarization ({square}) or MTOC reorientation ({blacksquare}). Graphs indicate data obtained from scoring at least 100 T cells per group and are representative of at least two independent experiments. Statistical analysis was performed using the Student t test or {chi}2. In A–C, p values between indicated groups are shown. In D, p values compared with appropriate medium only control are: {dagger}, p < 0.01; {ddagger}, p < 0.001.

 
We next performed dose-response experiments, to ascertain whether MTOC reorientation requires a higher threshold of TCR signaling than Ag-specific F-actin polarization. Ag-specific MTOC reorientation occurred in a dose-dependent manner, the responder T cell frequency being ~40% of maximum at 10 µg/ml. In contrast, 10 µg/ml OVA was sufficient to drive an almost maximal F-actin response (Fig. 2D). The observation that MTOC reorientation required a greater availability of MHC class II-peptide complexes raised the possibility that Leishmania infection limited the availability of MHC-peptide complexes at the cell surface.

The generation of class II-peptide complexes is not inhibited by L. donovani infection

The capacity of Leishmania-infected macrophages to process exogenous Ags and express class II is controversial (14, 15). Under our experimental conditions, we observed that both surface and intracellular class II molecules were abundant in Leishmania-infected BMM{phi} (Fig. 3, A–D), confirming our previous studies which demonstrated that class II biosynthesis and bulk traffic to the plasma membrane are not inhibited by L. donovani infection in these cells (35). Unfortunately, no reagents are available to directly evaluate H2Ad-OVA323–339 complex formation at the single-cell level. Therefore, we pulsed BMM{phi} from congenic BALB.K mice with HEL, and evaluated expression of surface H2Ak-HEL46–61 complexes using the complex-specific mAb C4H3 (37). As shown in Fig. 3E, assembly and delivery of H2Ak-HEL46–61 to the macrophage plasma membrane was relatively inefficient in BMM{phi}. Nevertheless, even under these conditions where small changes in processing efficiency should be readily observable, L. donovani infection does not significantly inhibit cell surface expression of H2Ak-HEL46–61 complexes (Fig. 3F). These data indicate that other mechanisms besides inefficient MHC expression or Ag processing must, therefore, limit the ability of cell surface MHC class II-peptide complexes on Leishmania-infected BMM{phi} to provide optimal TCR stimulation.



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  		FIGURE 3. Expression of MHC class II and of class II-peptide complexes is not inhibited by L. donovani infection. Confocal (A) and corresponding Nomarsky (B) image of infected BMM{phi} showing abundant surface and intracellular MHC class II. Arrowheads indicate L. donovani amastigotes. Flow cytometric analysis of uninfected (C) and L. donovani-infected (D) BALB.K-derived BMM{phi} stained with anti-H2Ak (shaded histogram) or isotype control (open histogram). FACS analysis of uninfected (E) and L. donovani-infected (F) BALB.K-derived BMM{phi} pulsed with (shaded histogram) or cultured without (open histogram) HEL and stained with mAb C4H3.

 
Efficient activation of effector T cells by BMM{phi} requires integrity of membrane rafts

Although uncharacterized in BMM{phi}, MHC class II molecules in other APC have been shown to be associated with various membrane microdomains, including lipid rafts (42, 43, 44, 45). Organization in such microdomains has been reported to be critical for optimal APC-T cell interactions (43, 46). Therefore, we examined whether lipid raft integrity was required for efficient effector T cell activation by BMM{phi}. BMM{phi} were pulsed with 10–100 µg/ml OVA or 5 µg/ml OVA323–339 and then treated with the raft-disrupting agent nystatin (47). Importantly, after fixation of nystatin-treated BMM{phi}, the drug was removed, so avoiding potential effects on the T cells themselves (48). As shown in Fig. 4A, OVA-pulsed BMM{phi} treated with nystatin were significantly inhibited in their ability to induce MTOC reorientation in T cells. In contrast, nystatin treatment had no effect on the response to exogenous OVA323–339. Given the greater efficiency of MHC class II-peptide complex formation after exposure to preformed peptide, these data support models whereby lipid rafts act to concentrate MHC class II-peptide complexes at the cell surface (43). Nystatin, though having a similar effect to L. donovani infection (Fig. 1), was nevertheless a more potent inhibitor of BMM{phi} function, and also affected Ag-dependent, but not Ag-independent, F-actin polarization in response to OVA (Fig. 4B).



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  		FIGURE 4. Disrupting the integrity of membrane rafts in BMM{phi} mimics the effect of L. donovani infection on MTOC reorientation. BMM{phi} were pulsed with OVA peptide (pep; 5 µg/ml OVA 323–339) or the indicated dose of OVA and then left untreated ({square}) or treated with 60 µg/ml nystatin ({blacksquare}). All cells were fixed and then allowed to form conjugates with DO.11 T cells. MTOC reorientation (A) and F-actin polarization (B) was then quantified. Graphical results indicate data obtained from scoring at least 150 T cells per group and are representative of two independent experiments. Statistical analysis was performed using the Student t test. Values of p compared with appropriate medium only control are: §, p < 0.05; {dagger}, p < 0.005; {ddagger}, p < 0.001.

 
The impact of Leishmania infection is dependent upon TCR signaling thresholds

The selective effect of L. donovani infection on T cell activation is consistent with a model whereby different T cell responses might require different thresholds of TCR engagement. This makes two predictions. First, increasing the availability of MHC class II-peptide complexes at the cell surface of L. donovani-infected BMM{phi} should rescue the MTOC response compromised by infection. As shown in Fig. 5A, when we examined MTOC reorientation in response to OVA323–339-pulsed cells, L. donovani-infected BMM{phi} were equally as efficient in activating T cells as uninfected macrophages. Thus, higher surface density of H2Ad-OVA323–339 overcomes the inhibitory effect of L. donovani on T cell activation (as it did on nystatin-treated cells, Fig. 4A). The second prediction was that effector responses requiring even higher thresholds of TCR signaling might be susceptible to inhibition by L. donovani, even at higher MHC class II-peptide complex density. Whereas F-actin and MTOC responses were readily detectable following stimulation with 10 µg/ml OVA (Fig. 2), detectable IFN-{gamma} synthesis by DO.11 T cells (Fig. 5, B–C) required 100 µg/ml OVA (Fig. 5D). L. donovani-infected BMM{phi}, as expected, were deficient in stimulating IFN-{gamma} after pulsing with OVA (~60% inhibition), but more significantly, inhibition was also seen following pulsing with OVA323–339 (~22%). Taken together, our results suggest that the functional deficit in L. donovani-infected BMM{phi} could result from suboptimal interactions between the TCR and MHC class II-peptide complexes expressed on infected macrophages.



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  		FIGURE 5. MTOC responses but not IFN-{gamma} production are recovered using peptide-pulsed L. donovani-infected macrophages. A, Uninfected ({blacksquare}) and L. donovani-infected macrophages ({square}) were left in medium or pulsed with OVA (100 µg/ml) or OVA323–339 (5 µg/ml) for 18 h before T cell conjugation. MTOC reorientation was scored as in Fig. 4. Confocal (B) and Nomarsky (C) image showing IFN-{gamma} production by DO.11 T cell after conjugation with an uninfected BMM{phi}. D, Analysis of the frequency of T cells bound to uninfected ({blacksquare}) or L. donovani-infected ({square}) BMM{phi} which make IFN-{gamma} in response to OVA or OVA323–339.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we have made the first formal description of the earliest events of T cell activation mediated by macrophages infected with L. donovani. Our data clearly show that the presence of L. donovani amastigotes can compromise the ability of macrophages to activate effector T cells when measured at the single-cell level. However, the degree to which various T cell responses are compromised reflects their requirements for distinct thresholds of TCR signaling. Our data also suggests that L. donovani infection fine tunes the capacity of BMM{phi} to present Ag by modulating the capacity of surface MHC class II-peptide complexes to engage the TCR.

To follow the early events after T cell activation, we have examined three parameters, namely F-actin polarization, MTOC reorientation, and IFN-{gamma} production. The role of F-actin during the process of T cell activation is still to be fully defined. However, published data (20) and our own (this study) indicate that significant F-actin polarization in T cells can occur in an Ag-independent manner, likely as a result of integrin-mediated interactions with APC. Nevertheless, TCR engagement increases the frequency of cells displaying F-actin polarization. Valensin et al. (24) have suggested that immediate TCR-proximal events including phosphorylation of CD3{zeta}-bound Vav regulate F-actin reorganization. Vav has previously been described to have guanine nucleotide exchange activity on the Rho family GTPase Rac1, a regulator of actin polymerization (49). It is proposed that these early Vav-mediated cytoskeletal events may underpin raft coalescence and sustained signaling in the immunological synapse (24). Similar step-wise activation of signaling cascades and supramolecular activation cluster formation have also recently been described by Kupfer and colleagues (25). In contrast to F-actin polarization, MTOC reorientation and effector cytokine production are strictly TCR-dependent events (Ref. 20 and this manuscript). MHC class II-peptide complex density plays an important role in driving signaling through the TCR (22, 43). Our data clearly support this concept, with differential threshold doses of Ag being required to observe Ag-dependent F-actin reorganization, MTOC polarization, and effector cytokine production. In some situations, costimulatory interactions, particularly those mediated through CD28 are believed to be important in modulating TCR-mediated signaling, and hence Ag dose thresholds for activation. Although L. donovani has been shown to down-regulate CD80 and CD86 expression (Ref. 40 and not shown), our model of BMM{phi}-effector T cell interaction is independent of costimulation mediated through CD28, alternate fixation-sensitive costimulatory receptor-ligand interactions (50), and of any requirement for APC membrane motility (51).

Collectively, our data argue that the availability of TCR ligands at the surface of infected macrophages is a limiting factor regulating cytokine production by effector T cells. Although previous studies have suggested that such limitations in Ag presentation might result from inhibition of Ag processing per se (4, 11, 14), in the current study we could find no evidence of this effect. The processing of exogenous OVA, measured by Ag-dependent F-actin polarization was normal (Fig. 1) and no deficit in the ability of Leishmania-infected BMM{phi} to process HEL and transport H2Ak-HEL46–61 complexes to the cell surface could be detected (Fig. 3). That complex formation and transport occurred with equal efficiency in infected and uninfected BMM{phi} was even more remarkable given the exceedingly low number of complexes formed, a situation which would have favored observation of other proposed mechanisms such as antigenic competition (4). It is now recognized, however, that MHC class II molecules on most APC are present in distinct membrane microdomains (43, 44, 45, 46). Lipid rafts may provide for clustering of MHC class II molecules irrespective of peptide specificity (43), whereas tetraspan microdomains allow selective organization of MHC class II-peptide complexes carrying a selective set of peptides (45). In both cases, the assembly of MHC-containing microdomains enhances the efficiency of Ag presentation, particularly at low Ag concentration. Our observation that effective Ag presentation by BMM{phi} is sensitive to nystatin treatment (Fig. 4) indicates that microdomains are required for optimal T cell activation under these experimental conditions. Disruption of microdomains could, therefore, be a mechanism by which infection with Leishmania lowers effective ligand availability without changing absolute levels of surface-expressed MHC class II-peptide complexes.

It is well-known that many intracellular pathogens, including Leishmania, release lipids, glycolipids, and glycophosphatidyl inositol anchored-glycoconjugates (52), and some of these molecules are widely distributed within the vesicular compartments of infected cells (52, 53, 54). The glycophosphoinositol anchors target such molecules to membrane microdomains (55, 56, 57, 58) and it is possible that their presence may disrupt the entry of or topography of microdomain-associated MHC class II-peptide complexes. In support of this hypothesis, the acquisition of raft-associated flotilin-1 by phagosomes has been shown to be inhibited by wild-type L. donovani promastigotes, but not by those deficient in the major surface glycolipid, lipophosphoglycan (59). Although lipophosphoglycan is expressed at negligible levels on the amastigotes of L. donovani used in our studies, this life cycle stage retains many other glycolipids, which may have similar function (60). The observation that exposing BMM{phi} to killed amastigotes can also lead to inhibition of MTOC responses further supports the notion that parasite constituents mediate this effect, rather than it being, for example, an active response on the part of the macrophage to viable infection. Future biochemical studies of the distribution of MHC class II molecules in infected BMM{phi} and BMM{phi} exposed to purified Leishmania glycoconjugates are now underway to test the hypothesis that Leishmania interferes with MHC class II organization in membrane microdomains.

Previous studies have indicated that Leishmania may limit Ag presentation in macrophages by restricting the availability of Ag for processing, for example, by expressing minimal levels of surface proteins and by restricting protein secretion (4, 11, 12). Our model of pulsing infected BMM{phi} with exogenous OVA is analogous to a situation where a small amount of protein Ag enters the endosomal compartment, either as a result of the death of occasional amastigotes, or through incomplete sequestration of amastigote-derived Ags. We suggest that Ag sequestration would be an even more effective strategy if the subsequent optimal display of cell surface MHC class II-peptide complexes were also impaired. It remains to be determined whether the various effects of Leishmania infection, noted here and elsewhere (10, 11, 12, 13, 14, 15, 16), also occur in dendritic cells infected with these parasites. Should this be the case, this may have additional implications for the priming of T cell responses during infection.

Finally, our data have important practical implications for the design of vaccines against leishmaniasis. The current emphasis on stimulating immunity with vaccines that couple high Ag dose with strong adjuvants would be expected to expand a broad repertoire of T cells with both low and high avidity TCR (61, 62), all competing for limited growth and survival factors and subsequently for space within the memory compartment (63, 64). Although this broad repertoire of T cells may be readily restimulated with Ag under optimal in vitro conditions, this may misrepresent their capacity to recognize infected macrophages. As low avidity T cells will require higher MHC class II-peptide complex density for their activation than that required by those with high avidity (62, 65, 66), the former may be unable to deliver focused macrophage activation in vivo. The priming of high avidity T cell responses is necessary for optimal antiviral immunity (66), and we would argue, on the basis of our present data, that vaccination strategies for leishmaniasis should similarly favor selective expansion of high avidity T cells. Indeed, our most successful attempts at vaccination against L. donovani have used low dose Ag administration in the absence of exogenous adjuvant (33). Furthermore, interventions predicted to enhance class II expression on Leishmania-infected macrophages (67, 68), or reduce the threshold for T cell activation (69), may prove to be effective alternatives for the treatment of leishmaniasis.


   Acknowledgments
 
We thank D. Gray, F. Powrie, P. Lane, H. Reiser, C. Reis e Sousa, and R. Germain for generously providing mice and reagents, the National Institutes of Health AIDS Research and Reference Program for rIL-2, and C. Engwerda for critical comments on this manuscript.


   Footnotes
 
1 This work was supported by the British Medical Research Council and The Wellcome Trust. Back

2 Address correspondence and reprint requests to Dr. Paul Kaye, Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT, U.K. E-mail address: paul.kaye{at}lshtm.ac.uk Back

3 Abbreviations used in this paper: MTOC, microtubule organizing center; BMM{phi}, bone marrow-derived macrophage; F-actin, filamentous actin; HEL, hen egg lysozyme; DAPI, 4',6'-diamidino-2-phenylindole. Back

Received for publication January 27, 2003. Accepted for publication October 9, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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