Brief Definitive Report |
Address correspondence to Terri M. Laufer, Dept. of Medicine, University of Pennsylvania, 753 BRB II/III, 421 Curie Blvd., Philadelphia, PA 19104. Phone: (215) 573-2975; Fax: (215) 573-7599; email: tlaufer{at}mail.med.upenn.edu
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Abstract |
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Key Words: Langerhans cells CD4 macrophages Th1 antigen presentation
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Introduction |
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MHC II+ DCs prime CD4+ Th1 cells to nominal antigens (5) and could fulfill this role during L. major infection (1). CD8+ and CD11b+ DCs can be infected in vitro (6), and T cell priming to an immunodominant L. major peptide is mediated by CD11b+ DCs (7). Transfer of L. majorinfected LCs can also confer resistance to susceptible strains (8). The DC subsets that initiate Th1 priming in vivo have not been delineated clearly.
The local accumulation of MHC II+ infected macrophages and their role in parasite lysis suggest that these cells are the central APC-mediating parasite control (3, 9). Production of IL-12, TNF, nitric oxide (NO), and IFN
by macrophages can contribute to Th1 polarization, effector Th1 CD4+ T cell maintenance, and parasite lysis (911). Macrophage secretion of these molecules can be facilitated by MHC II peptideTCR and CD40CD40L interactions (12, 13), but noncognate mechanisms such as Toll-like receptor signaling, TNF
, and IFN
also elicit cytokine secretion and activation (14). Thus, the relative requirement of MHC II expression within different DC subsets and macrophages has not been established.
To study the cognate interactions that mediate control of L. major, we took advantage of the CD11c/Aßb transgenic mouse model in which MHC II expression is restricted to CD11b+ and CD8+ DCs (5). We report that CD11c/Aßb mice develop normal immunity to the intracellular parasite, L. major, and control this subcutaneous infection, indicating that antigen presentation by CD11b+ and CD8
+ DCs is sufficient for Th1 differentiation and effector functions throughout the primary response.
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Materials and Methods |
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Contact Sensitization and LC Isolation.
Mice were anesthetized, shaved, and painted on the back with 30 µl FITC (Sigma-Aldrich) diluted in 1:1 acetone/dibutyl acetate (Sigma-Aldrich) as described previously (15). 1824 h after application of FITC, brachial, inguinal, and auricular LNs were digested at room temperature in 0.8 mg/ml collagenase type IV (Worthington Biochemicals) and 0.05% DNase I (Sigma-Aldrich) for 3045 min. CD11c+ cells were purified using CD11c MACS beads (Miltenyi Biotech) on paramagnetic AUTOMACS columns according to the manufacturer's instructions. CD11c+ cell purity ranged from 85 to 95%.
Flow Cytometry.
Single cell suspensions were blocked with anti-FcRII/III (24G2; American Type Culture Collection). Staining of APCs was performed in PBS with 2% BSA, 2 mM EDTA, 1 µg/ml mouse IgG (Sigma-Aldrich), and 1 µg/ml rat IgG (Sigma-Aldrich). All conjugated antibodies used for staining were obtained from BD Biosciences. Intracellular staining was performed on 1% PFA fixed samples, which were permeabilized with 2% BSA and 0.02% saponin in PBS. For intracellular IFN staining, LN cells were first stimulated with 50 ng/ml PMA and 500 ng/ml ionomycin in the presence of 2 nM monensin for 5 h at 37°C. Samples were analyzed on a FACSCaliburTM (BD Biosciences) using CELLQuestTM software. All dot plots have a log axis of 100104.
Polyclonal T Cell Purification.
B6.SJL spleen and LN suspensions were incubated with supernatants of 24G2, RA3, M1/70, and M5/114 (American Type Culture Collection) followed by goat antirat IgG magnetic beads (Polysciences, Inc.) and cells were removed by attaching to a magnetic stand (Bio-Mag; Polysciences, Inc.).
L. major Infections and Analysis.
Purified CD45.1+ CD4+ T cell suspensions were transferred i.v. into hosts. 1 d later, the hind footpad was infected with 5 x 105 stationary phase L. major promastigotes (MHOM/IL/80/Friedlin) grown in Grace's media as described previously (16). Footpad swelling was measured with a digital caliper and reported as the difference between infected and uninfected hind footpads. Standard deviation represents the difference in measurements within individual mice of the same genotype. Mice with swelling >5 mm were killed.
4, 7, or 9 wk after infection, infected footpads were processed for parasite counts (16) or immunohistochemistry (5). Antibodies GK1.5, M5/114, N418 (American Type Culture Collection), inducible NO synthase (iNOS)FITC, and IFN (BD Biosciences) were used. FITC-stained slides were visualized on an IMT-2 fluorescence microscope (Olympus) and analyzed using SPOT software (HiTech).
Splenic CD4+ T cells were purified using CD4 MACS beads (Miltenyi Biotech) on AUTOMACS columns. C57BL/6 T-depleted splenocytes were prepared by low-toxicity complement depletion (Cedarlane) with anti-Thy1 (MMT1) antibodies. 106 CD4+ T cells were cultured with 5 x 106 C57Bl/6 T-depleted splenocytes and 10 µg/ml soluble L. major antigen (16) for 3 d. Supernatants were analyzed by sandwich ELISA for IFN production.
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Results and Discussion |
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MHC II Expression in CD11c/Aßb Mice.
First, we determined whether APC subsets from CD11c/Aßb mice maintained their MHC IInegative phenotype after exposure to inflammatory signals in vivo. LCs migrating out of skin explants lacked MHC II expression in CD11c/Aßb mice (5). To examine LC expression during inflammation, we painted the ears and dorsal skin of mice with FITC diluted in irritant. In response to the stimulus, FITC-painted epidermal DCs migrated into the draining LNs, and were detected within 1648 h (reference 15 and unpublished data). FITC-painted cells only migrated into skin-draining LNs, indicating specific painting of LCs (unpublished data). We analyzed FITC-negative and FITC-positive DCs in skin-draining LNs for MHC II expression (Fig. 1 A). FITC-painted LCs from Aßb+/- mice were MHC II bright; whereas, FITC-painted LCs from CD11c/Aßb mice remained MHC II negative. In contrast, FITC-negative DCs from Aßb+/- and CD11c/Aßb mice (including CD11b+ and CD8+ cells) stained MHC II positive. Thus, CD11b+ and CD8
+ LN DCs from CD11c/Aßb mice, but not LCs, are MHC II positive during inflammation.
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Macrophages and B cells in the CD11c/Aßb mouse model remain MHC II negative after in vitro exposure to IFN or LPS (5). Macrophages from L. majorinfected Aßb-/- and CD11c/Aßb mice also lacked surface MHC II, but Aßb+/- macrophages were MHC II positive (Fig. 1 B). Similarly, Aßb+/- B cells had MHC II expression; whereas, Aßb-/- and CD11c/Aßb B cells were MHC II deficient (Fig. 1 C).
Finally, it was important to establish that MHC II expression at the site of infection mirrored that in the LNs. Diffuse I-Ab staining was observed in Aßb+/- footpads, where MHC II expression was present in macrophages, CD11c+ DCs, and parenchymal cells (Fig. 4 C and not depicted; reference 3). In contrast, CD11c/Aßb lesions had MHC II expression limited to isolated CD11c+ foci (Fig. 4 C and not depicted). Thus, antigen presentation in CD11c/Aßb mice during L. major infection is restricted to CD8+ and CD11b+ DCs both in the LN and at the primary site of infection.
Control of L. major.
CD11c/Aßb mice lack MHC II expression on cortical thymic epithelium and have no MHC IIrestricted CD4+ T cells (5). However, adoptive transfers of naive polyclonal CD4+ T cells into SCID and RAG-deficient mice restore control of L. major infections (18, 19). Therefore, we transferred 1520 x 106 polyclonal CD45.1+ T cells into Aßb-/-, Aßb+/-, or CD11c/Aßb mice 1 d before infection. TCR-/- mice were also infected because they have wild-type expression of I-Ab but, like CD11c/Aßb mice, would depend on transferred cells for parasite control. Mice were infected in the footpad with 5 x 105 stationary phase promastigotes on day 1, and footpad swelling was monitored weekly (Fig. 2 A). In Aßb+/- mice, lesion size peaked at week 5 and resolved by week 8. In contrast, Aßb-/- mice developed large nonhealing lesions requiring euthanasia between weeks 6 and 9. Parasite titers were consistent with lesion size (Fig. 2 B). Thus, control of parasite growth and lesion resolution was MHC II dependent.
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L. majordependent Th1 Differentiation.
To examine Th1 differentiation in response to L. major, IFN production was assayed. First, CD4 T cells from draining (popliteal) or distant (cervical) LNs were harvested 4 or 9 wk after infection, and IFN
production was assayed after restimulation with PMA and ionomycin (Fig. 3 A). Distant cervical LNs served as a control for the absence of L. majorspecific responses. CD45.1+ cells (right quadrants) are transferred MHC IIrestricted CD4+ T cells; the CD45.1- CD4+ T cells are endogenous cells (left quadrants). Aßb+/- mice have endogeneous MHC IIrestricted CD4 T cells, which produced most of the IFN
in the popliteal LNs. However, more CD4 T cells in the popliteal LNs produced IFN
than at distant sites. In contrast, MHC IIrestricted transferred CD4+ T cells did not accumulate in the draining LNs of Aßb-/- mice (46% of the transferred CD45.1+ cells produced IFN
at this site, a proportion comparable to the staining in the cervical LNs). Endogenous MHC IIindependent CD4+ cells (probably NKT cells) do accumulate at the site of infection in Aßb-/- mice, but cannot control the pathogen (4, 20, 21).
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L. majordependent Phagocyte Activation.
Parasite control depends on activation of phagocytes (2). Thus, either macrophage activation in CD11c/Aßb mice is noncognate, or MHC class IIpositive DCs can subsume functions normally attributed to macrophages. To analyze macrophage activation, we examined CD40 up-regulation and iNOS expression in macrophages harvested from popliteal LNs. Macrophages accumulated in the draining LNs of Aßb+/-, CD11c/Aßb, and TCR-/- mice, but not I-Aßb-/- mice (unpublished data). However, surface expression of CD40 staining was equivalent in all genotypes (Fig. 4 A). Thus, CD40 expression is independent of either Th1 effectors or cognate antigen presentation.
iNOS-dependent NO production also contributes to L. major control (2). Expression of iNOS in popliteal LN macrophages was only detectable in Aßb+/-, CD11c/Aßb, and TCR-/- macrophages (Fig. 4 B). Macrophages in Aßb+/- and TCR
-/- mice contained more iNOS than those in CD11c/Aßb mice (Fig. 4 B), suggesting a cognate component to regulation of the enzyme, but this had no effect on parasite loads at weeks 4, 7, and 9 (Fig. 2 B and not depicted). Immunofluorescence of the footpad revealed that iNOS was also present in the infection site of Aßb+/-, CD11c/Aßb, and TCR
-/-, but not Aßb-/- mice (Fig. 4 E). Thus, cognate macrophageCD4 T cell interactions are not required for either CD4+ T cell recruitment to inflamed tissues or macrophage activation and effector functions.
One interpretation of our results is that IFN production by differentiated effector CD4+ T cells at the site of infection is antigen dependent, and relies on CD11b+ and CD8
+ DCs. Indeed, we detected recruitment of CD11c+ cells to local sites of infection. Therefore, the role of DCs in the primary immune response could extend beyond initiation of CD4+ T cell responses to control CD4+ T cell functions during the effector phase. Alternatively, IFN
secretion from activated CD4+ T cells could occur in the absence of cognate antigen presentation in vivo. In this scenario, MHC IITCR interactions would be required for priming, but activated effector CD4+ T cells could secrete IFN
without further TCR triggering. This would imply that the effector cytokine delivery of the CD4+ T cell responses might be less specific than previously thought.
Finally, control of L. major infection also requires phagocyte activation and the killing of intracellular amastigotes (2). In CD11c/Aßb mice, MHC IIdeficient macrophages must respond to IFN and/or CD40CD40L interactions without cognate T cell interactions. Thus, pathogen clearance functions attributed to macrophages, such as NO production, phagocytosis, and lysis of infectious organisms (9), lack the specificity requirements of antigen presentation. Our results imply that the immune system can avoid pathogens' attempts to block antigen presentation within the infected macrophage because antigen presentation by this cell type is not a requirement for activation or parasite lysis.
In conclusion, we have demonstrated the sufficiency of CD11b and CD8+ DC antigen presentation in the initiation and effector phases of Th1 immunity to L. major. Our findings suggest that antigen presentation by macrophages, keratinocytes, pDCs, and LCs are not required for either differentiation of Th1 CD4+ T cells responding to L. major or the effector phases that follow. It will be interesting to determine if other APCs play a more important role in secondary immune responses against L. major because memory CD4+ T cells survey tissues where resident L. majorinfected macrophages and LCs might be more important APCs.
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Acknowledgments |
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This work was supported by a Grant-in-Aid from the Pennsylvania/Delaware affiliate of the American Heart Association (T.M. Laufer) and the National Institutes of Health (AI 35914 to P. Scott).
Submitted: 13 May 2003
Accepted: 18 December 2003
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References |
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