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The Journal of Immunology, 2002, 168: 3992-4000.
Copyright © 2002 by The American Association of Immunologists

CD8+ T Cells Are Required for Primary Immunity in C57BL/6 Mice Following Low-Dose, Intradermal Challenge with Leishmania major

Yasmine Belkaid*, Esther Von Stebut{dagger}, Susana Mendez*, Rosalia Lira*, Elisabet Caler*, Sylvie Bertholet*, Mark C. Udey{dagger} and David Sacks1,*

* Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, and {dagger} Dermatology Branch, Division of Clinical Sciences, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Standard murine models of cutaneous leishmaniasis, involving s.c. inoculation of large numbers of Leishmania major promastigotes, have not supported an essential role for CD8+ T cells in the control of primary infection. Recently, a L. major model combining two main features of natural transmission, low parasite dose and inoculation into a dermal site, has been established in resistant C57BL/6 mice. In the present studies, C57BL/6 mice with CD8+ T cell deficiencies, including CD8-/- and CD8-depleted mice, failed to control the growth of L. major following inoculation of 100 metacyclic promastigotes into the ear dermis. The resulting dermal pathology was minor and delayed. Lesion formation in wild-type mice was coincident with the killing of parasites in the inoculation site. Both events were associated with the accumulation of CD8+ T lymphocytes in the skin and with the capacity of CD8+ T cells recovered from draining lymph nodes or infected dermis to release IFN-{gamma} following coculture with infected dendritic cells. Reconstitution of resistance to L. major in RAG-/- mice using T cells from naive donors was optimal when both CD4+ and CD8+ T cells were transferred. Primed CD8+ T lymphocytes obtained from C57BL/6 mice during the acute stage of infection were able to mediate both pathology and immunity when transferred alone. The low dose, intradermal challenge model reveals that CD8+ T cells play an essential role in both pathogenesis of and immunity to primary infection with L. major in the skin.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Infection of mice with the protozoan parasite Leishmania major has been extensively used to define the factors controlling the differentiation of CD4+ T lymphocyte subsets in vivo. Using the standard challenge model involving s.c. injection of high doses (105–107) of parasites in the footpad, and depending on the genotype of the mouse, L. major induces a Th1- or Th2-predominant response that respectively controls or promotes intracellular growth of the parasite in the inoculation site. Acquired resistance in this model has been shown to require CD4+ T cells, activated in an IL-12-driven and CD40L-dependent manner, for the production of high levels of IFN-{gamma} that induce NO-dependent killing by infected macrophages (1, 2, 3, 4, 5, 6, 7, 8, 9, 10). Although CD8+ T cells were shown to be essential to immunity to reinfection in resistant mice that healed their primary lesion (11, 12, 13, 14), the role of CD8+ T cells in the control of primary infection remains unclear. A higher frequency of parasite-specific CD8+ T cells was found in resistant (C57BL/6) compared with susceptible (BALB/c) mice (2, 11). Furthermore, administration of anti-CD8 Ab to resistant mice exacerbated infection, although treated mice were still able to ultimately heal (2). C57BL/6 mice with {beta}2-microglobulin or CD8 deficiencies also maintained their ability to heal, strongly indicating that CD8+ T cells are not required for control of primary infection (15, 16, 17).

In contrast to the conventional challenge models used in the studies described above, in natural transmission a low number (<1000) of metacyclic promastigotes (18) is delivered by the sand fly into the dermis of the mammalian host. We previously established a model of infection that combines two main features of natural transmission: low dose (100 metacyclic promastigotes) and cutaneous inoculation (the mouse ear dermis) (19). The model revealed two phases in the pathogenesis of dermal leishmaniasis that had not been distinguished previously: a remarkably silent phase, lasting 4–5 wk, favoring the uncontrolled growth of parasites in the skin without formation of a lesion, followed by development of a dermal lesion that is coincident with the onset of immunity and killing of the parasite in the inoculation site. A role for CD4+ T cells in both pathogenesis and immunity was confirmed in the natural infection model.

In the present studies, CD8+ T cells are reexamined in the context of the low dose, intradermal challenge model, including their homing to and accumulation in the skin, their capacity to produce IFN-{gamma} in response to parasite Ag, their role in the pathogenesis of dermal lesions, and their requirement for the control of infection and the resolution of dermal disease.


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

C57BL/6 mice were purchased from the Division of Cancer Treatment, National Cancer Institute (Frederick, MD). RAG1-/- mice (backcrossed for 10 generations to C57BL/6) were purchased from Taconic Farms (Germantown, NY). CD8{alpha}-/- mice (backcrossed for at least five generations to C57BL/6) were kindly provided by A. Singer (National Cancer Institute, National Institutes of Health, Bethesda, MD). All mice were maintained in a National Institute of Allergy and Infectious Diseases animal care facility under specific pathogen-free conditions.

Parasite preparation and intradermal inoculation

L. major clone V1 (MHOM/IL/80/Friedlin) was cultured in Medium 199 with 20% heat-inactivated FBS (HyClone Laboratories, Logan, UT), 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM L-glutamine, 40 mM HEPES, 0.1 mM adenine (in 50 mM HEPES), 5 mg/ml hemin (in 50% triethanolamine), and 1 mg/ml 6-biotin (M199/S). Infective-stage metacyclic promastigotes of L. major were isolated from stationary cultures (4–5 days old) by negative selection using peanut agglutinin (Vector Laboratories, Burlingame, CA). Metacyclic promastigotes (100) were inoculated intradermally into the ear dermis using a 27.5-gauge needle in a volume of ~5 µl. The evolution of the lesion was monitored by measuring the diameter of the induration of the ear lesion with a direct-reading vernier caliper (Thomas Scientific, Swedesboro, NJ).

CD8+ cell depletion

Mice were inoculated i.p. with 1 mg of rat mAb anti-CD8 (2.43) or rat isotype control (GL113), purified from ascites by ammonium sulfate precipitation, at the time of the challenge and weekly thereafter until termination of the experiments.

Estimation of parasite load

Parasite titrations were performed as previously described (19). The two sheets of infected ears were separated, deposited dermal side down in DMEM containing 100 U/ml penicillin, 100 µg/ml streptomycin, and 125 U/ml collagenase A (Sigma-Aldrich, St. Louis, MO), and incubated for 2 h at 37°C. Sheets were subsequently cut into small pieces and homogenized using a Teflon-coated microtissue grinder in a microfuge tube containing 100 µl of M199/S. Tissue homogenates were filtered using a 70-µm cell strainer (Falcon Products, St. Louis, MO) and serially diluted in 96-well flat-bottom microtiter plates containing biphasic medium prepared using 50 µl of NNN medium containing 30% of defibrinated rabbit blood overlaid with 50 µl of M199/S. The number of viable parasites in each sample was determined from the highest dilution at which promastigotes could be grown out after 7 days of incubation at 26°C.

Analysis of intradermal lymphocytes

To characterize leukocytes in the inoculation sites, the ears were collected, and the ventral and dorsal dermal sheets were separated and incubated dermal side down on RPMI 1640, NaHCO3, penicillin/streptomycin/gentamicin, containing 125 U/ml collagenase A (Sigma-Aldrich) for 2 h. The dermal sheets from three to five animals were pooled, cut into small pieces, and filtered through a 70-µm nylon cell strainer (BD Biosciences, Mountain View, CA) before being washed twice in RPMI 1640, NaHCO3, penicillin/streptomycin/gentamicin, 10% FCS, and 0.05% DNase I (Sigma-Aldrich). Cells were analyzed by flow cytometry for surface markers and/or cytoplasmic staining for IFN-{gamma}, as described below.

Preparation of APC, lymphocytes, and coculture

In some experiments, unfractionated lymph node (LN)2 cells, purified lymphocyte subsets, or dermal cells were incubated with fetal skin-derived dendritic cells (FSDDC) prepared as previously described (20). Briefly, skin cells from day 16 fetal C57BL/6 or BALB/c mice were cultured in GM-CSF- and M-CSF-supplemented media and, after 2 wk, dendritic cell (DC) aggregates were isolated by serial 1-g sedimentation. DC aggregates were dissociated in trypsin/EDTA (0.25%/0.1 mM) as necessary to allow accurate determination of cell number. FSDDC aggregates were subcultured in growth media (5% FBS, GM-CSF- and M-CSF-supplemented RPMI 1640) in 24-well plates at 2 x 105 cells in 1 ml/well. L. major amastigotes, purified from footpad lesions and opsonized by incubation for 30 min at 37°C in 5% fresh normal mouse serum, were added at approximately five organisms per cell. FSDDC aggregates were completely dissociated in calcium- and magnesium-free HBSS containing 1 mM EDTA (30 min at 37°C). In some experiments, DC were generated from bone marrow in the presence of GM-CSF (BMDC), as previously described (21). Bone marrow-derived macrophages (BMDM) were cultured with 30% L929 supernatant for 5 days, and nonadherent cells were recovered and incubated for 18 h in 2.5% L929 supernatant. Adherent cells were recovered and infected for 18 h with serum-opsonized amastigotes (five amastigotes per macrophage). Aliquots of cells were prepared in a cytospin and stained using Diff Quick (Dade Behring, Dudinger, Switzerland) to estimate the level of infection. CD8+ and CD4+ T cells were isolated by positive selection using magnetic beads (Miltenyi Biotec, Bergisch Gladbach, Germany). The purity of CD8+ or CD4+ T lymphocytes was >95%. Unfractionated LN or dermal cells were resuspended in complete RPMI 1640 at 2.5 x 106 cells/ml, and 1 ml of the cell suspension was incubated with or without soluble Leishmania Ag (SLA; 25 µg/ml) or with infected or uninfected FSDDC or BMDC at a ratio of 5:1 in 24-well plates at 37°C in 5% CO2 for 18 h. The cells were harvested and analyzed for surface markers and intracytoplasmic staining for IFN-{gamma}. Purified CD8+ or CD4+ T cells were cocultured with uninfected, infected, or Ag-loaded BMDM or FSDDC at a ratio of 5:1 (5 x 105 cells total) in U-bottom 96-well plates and incubated at 37°C in 5% CO2 for 24 h. In some experiments, unfractionated LN cells were recovered and incubated for 48 h with SLA (25 µg/ml), and IFN-{gamma} or IL-4 in culture supernatants was quantitated by ELISA, as previously described (22).

Immunolabeling and flow cytometry

Before staining, LN or dermal cells were incubated with an anti-Fc III/II receptor (BD PharMingen, San Diego, CA) mAb and 10% normal mouse serum in PBS containing 0.1% BSA and 0.01% NaN3. The lymphocytes were identified by characteristic size (forward light scatter (FSC)) and granularity (side light scatter (SSC)), in combination with anti-TCR{beta} chain (H57-597, FITC conjugated; BD PharMingen) and anti-CD4 or anti-CD8 (CyChrome conjugated) surface staining. The staining of surface and intracytoplasmic markers was performed sequentially: the cells were stained first for their surface markers (TCR{beta} chain receptor, CD8 or CD4), followed by a permeabilization step and staining with anti-IFN-{gamma} (XNG1.2; BD PharMingen). The isotype controls used (all from BD PharMingen) were rat IgG2b (A95-1), rat IgG2a (R35-95), and hamster IgG, group 2 (Ha4/8). For each sample, between 200,000 and 400,000 cells were analyzed. The data were collected and analyzed using CellQuest software and a FACSCalibur flow cytometer (BD Biosciences).

Reconstitution of C57BL/6 RAG-/- mice

Naive (4 x 106) or primed (2 x 106) CD4+ and/or CD8+ T cells were injected i.v. in 300 µl of PBS into RAG-/- mice. Unreconstituted and reconstituted RAG-/- mice were challenged the same day with 103 L. major metacyclic promastigotes in the dermis of both ears. Mice reconstituted with CD8+ T cells were also inoculated the same day and 1 wk later with 1 mg of anti-CD4 (GK1.5). Parasite load and lesion size were followed, as previously described. Between 4 and 12 wk following infection, cells from the inoculation site and the draining LN were prepared as described above and incubated for 2 h in the presence of 10 µg/ml anti-CD28 (37.51; BD PharMingen), 5 ng/ml IL-2 (Endogen, Woburn, MA), and 25 µg/ml SLA, followed by a 4-h incubation with brefeldin A (Sigma-Aldrich). Cells were analyzed by FACS, as described.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Parasite loads and dermal lesions in C57BL/6, CD8-/-, and CD8-depleted mice following inoculation of 100 L. major metacyclic promastigotes in the ear

To characterize the role of CD8+ T cells in the control of primary infection due to L. major, 100 metacyclic promastigotes were inoculated into the ear dermis (both ears) of wild-type C57BL/6 mice, CD8-/-, or CD8-depleted C57BL/6 mice. The number of parasites in the inoculation sites was determined at different time points, and the evolution of the lesions (measured by nodule diameter) was monitored every week over a period of 17–24 wk (Fig. 1Go). In C57BL/6 wild-type mice or mice injected weekly with the anti-CD8 isotype control, the parasites grew to maximal numbers in the skin during the first 4–5 wk without inducing lesions (19). Control of parasite growth was apparent during wk 5 and 6, and was accompanied by the development of a small nodule that completely resolved by 9–10 wk postinjection (Fig. 1Go). There were no significant differences between the control groups and the various CD8-deficient groups in their dermal parasite loads up to wk 5. By 6–8 wk, when >95% of the parasites had been cleared from the site in the wild-type or control-treated mice, parasites continued to grow in the CD8-deficient mice such that by wk 10, the ears of CD8-/-- and anti-CD8-treated mice contained, respectively, 2 and 3.3 log-fold more parasites compared with their respective controls (Fig. 1Go). To confirm that this effect was not transient, all experiments were conducted over a period of 4–5 mo. No reduction in the total number of parasites in the inoculation site was observed in the CD8-deficient mice over this period. Parasite loads in the CD8-/- and the CD8-depleted mice stabilized at 106 and 108 parasites per ear, respectively, at about wk 16. These high parasite loads were associated with relatively minor dermal pathology, characterized by nonulcerative nodules that failed to heal (Fig. 1Go). The persistence of variable numbers of parasites in the skin of the wild-type and control-treated mice following healing of their dermal lesions has been reported previously, and is dependent on the production of IL-10 in the chronic site (23). The CD8 depletion was associated with a significant reduction in the amount of IFN-{gamma} produced by LN cells in response to SLA 3.5 and 8 wk after infection (Table IGo) compared with the isotype control. The production of IL-4 was slightly increased 8 wk postchallenge in anti-CD8-treated mice compared with the isotype control (305 ± 10 vs 123 ± 7).



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  		FIGURE 1. Number of parasites per ear ({blacksquare} and {square}) and diameter of induration (• and {circ}) following intradermal inoculation of 100 L. major metacyclics in A, C57BL/6 mice ({square} and {circ}) or in CD8-/- mice ({blacksquare} and •), and B, CD8-depleted ({blacksquare} and •) or isotype-treated ({square} and {circ}) C57BL/6 mice. Values represent mean induration in mm ± SD, five mice per group, and geometric mean parasite number per ear ± SD, four mice and eight ears per group.

 

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  		Table I. Effect of anti-CD8 depletion on Leishmania-specific cytokines production by LN cells draining the ear 3.5 and 8 wk postinfection

 
T cell activation in the draining LN of C57BL/6 mice following low-dose, intradermal challenge

Naive T cells typically express high levels of CD62L (L-selectin), low levels of CD25 (IL-2R{beta} chain), and low levels of the early activation marker CD69. We followed expression of these markers on CD4+ and CD8+ T cells present in draining LN during the course of the infection in the ear dermis (Fig. 2Go). The proportion of CD4+ T cells expressing an activated phenotype increased as early as 1 wk postinfection, with CD25+ cells increasing from 13 to 25%, CD62Llow from 9.5 to 18%, and CD69+ from 13 to 17%. Expression of these activation markers on CD8+ T cells was delayed until 4–5 wk, just before the onset of lesion formation and killing of parasites in the inoculation site.



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  		FIGURE 2. FACS analysis of surface activation markers expressed by CD4+ or CD8+ T cells in the draining LN following intradermal inoculation of 100 L. major metacyclics in C57BL/6 mice. For each marker, the histograms of gated TCR{beta}+/CD4+ or TCR{beta}+/CD8+ cells are shown. For CD25 and CD69, the numbers represent the percentage of cells with FL1 signals greater than that detected with the appropriate isotype control. For CD62, the numbers represent the percentage of cells that stained negative for the marker based on a comparison with isotype control. The draining LN from four mice were pooled for each time point. The experiment is representative of two separate experiments.

 
Recruitment of CD8+ and CD4+ T lymphocytes to the inoculation site

The L. major-loaded dermis was analyzed for the presence of cells bearing TCR{beta} and either CD4 or CD8 by two-color FACS analysis of cells extracted from collagenase-treated skin (Fig. 3Go). A small number of CD4+ T lymphocytes (4.5 x 103 cells/ear), but no CD8+ T cells were found in the ear dermis of naive mice. These steady-state conditions were maintained until wk 4, at which time there was a substantial increase in CD4+ T cells to 4.2 x 104/ear, and the onset of CD8+ T cell recruitment to the site (7.2 x 103). At 6 wk, their cell numbers increased to 8.9 x 104 CD4+ and 2.8 x 104 CD8+ T cells/ear dermis.



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  		FIGURE 3. Dot plots of TCR{beta}+/CD4+ and TCR{beta}+/CD8+ cells present in the ear dermis following intradermal inoculation of 100 L. major metacyclic promastigotes in C57BL/6 mice. The numbers represent the absolute number of cells per ear positive for each of the two markers. Ten ears from five mice were pooled for each time point. The experiment is representative of three separate experiments.

 
APC requirements for Ag-specific IFN-{gamma} production by CD4+ and CD8+ T cells in draining LN and dermis

Because IFN-{gamma} production remains an absolute requirement for acquired immunity in the natural-challenge model (19), we examined the ability of CD4+ and CD8+ T cells present in draining LN 5 wk postinfection to produce IFN-{gamma} in response to SLA (Fig. 4Go). An increased frequency (1.7%) of CD4+ T cells, but not of CD8+ T cells able to produce IFN-{gamma} was observed. Because the APC and exogenous Ag present in the whole LN cultures may not have optimized the conditions necessary to reveal activation of CD8+ T cells for production of IFN-{gamma}, FSDDC, which have been shown to up-regulate MHC class I, class II costimulatory molecules, and IL-12 p40 in response to uptake of L. major amastigotes (24), were loaded with SLA or infected with amastigotes and added to the cultures. CD4+ T cells were able to release IFN-{gamma} when FSDDC alone were added to the LN cultures (1.8%), perhaps due to the presence of a small amount of Ag released by the infected LN cells. No IFN-{gamma}-producing cells were seen when FSDDC were incubated with LN cells from uninfected mice (data not shown). The percentage of IFN-{gamma}-producing CD4+ T cells was slightly increased when the LN cells from infected mice were incubated with FSDDC loaded with SLA (1.8%) or live parasites (2.3%). In contrast, only FSDDC infected with live L. major induced substantial production of IFN-{gamma} by CD8+ T cells (3.2%).



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  		FIGURE 4. Intracytoplasmic staining for IFN-{gamma} in CD8+ or CD4+ T cells from draining LN (A) or dermis (B) 6 wk following intradermal inoculation of 100 L. major metacyclic promastigotes in C57BL/6 mice. Whole LN cells were cultured alone or with SLA, SLA plus FSDDC, or FSDDC infected with L. major amastigotes. Dermal cells were cultured with noninfected or infected BMDC. Events were initially gated on size and granulosity, then on TCR{beta} expression. The numbers represent the percentage of CD4+ or CD8+ T cells positive for IFN-{gamma}, based on comparison with FL2 isotype control. The experiment is representative of two separate experiments.

 
To determine whether the CD8+ T cells extracted from skin were also able to release Ag-specific IFN-{gamma}, dermal cells were purified from 6-wk-infected mice and incubated with either L. major infected or noninfected BMDC. A small percentage of CD4+ (0.7%) or CD8+ T cells (0.2%) produced IFN-{gamma} when exposed to noninfected BMDC. When exposed to infected DC, a high percentage of both CD4+ (18%) and CD8+ T cells (4.5%) was able to produce IFN-{gamma} (Fig. 4GoB).

To confirm that the primed CD8+ T cells can secrete IFN-{gamma} and to investigate further their selective reactivation requirements, positively selected CD4+ and CD8+ cells prepared from LN of C57BL/6 mice 5 wk postchallenge were incubated with infected or SLA-pulsed macrophages or FSDDC and assayed for IFN-{gamma} release by ELISA (Fig. 5Go). Although CD4+ T cells released high and comparable amounts of IFN-{gamma} regardless of the source of Ag or APC used for their activation, CD8+ T cells released IFN-{gamma} only when incubated with Ag-loaded FSDDC (5.2 ng/ml) or with FSDCC infected with L. major amastigotes (10 ng/ml). In addition to BMDM, resident peritoneal macrophages or inflammatory macrophages obtained from nonimmune granulomas also failed to activate CD8+ T cells for IFN-{gamma} production (data not shown).



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  		FIGURE 5. IFN-{gamma} production by CD8+ or CD4+ T cells from draining LN 6 wk following intradermal inoculation of 100 L. major metacyclic promastigotes in C57BL/6 mice. CD4+ ({square}) and CD8+ ({blacksquare}) T cells were purified and incubated with noninfected, infected, or SLA-loaded FSDDC or BMDM. IFN-{gamma} levels in culture supernatants were determined by ELISA after 24-h incubation. The data shown are the mean ± SD of three replicates, and the experiment is representative of three separate experiments.

 
Immunity in RAG-/- mice reconstituted with naive CD8+ and/or CD4+ T cells

To assess the effector capacity of CD8+ lymphocytes in vivo, and to determine whether these cells could mediate protection and pathology, C57BL/6 RAG-/- mice, deficient in the recombinase-activating gene required for development of mature B or T cells, were reconstituted with CD4+ and/or CD8+ T cells recovered from naive wild-type mice. The recipient and unreconstituted mice were challenged in the ear with 1000 metacyclic promastigotes. The transferred CD8+ cells did not contain CD8+ DC, as assessed by the absence of MHC class II-positive cells. The purified CD8+ population did, however, contain small numbers (<1%) of CD4+ lymphocytes, which in an initial study led to the accumulation of large numbers of CD4+ T cells in inoculated ears (55% of CD3+ cells) and in draining LN (30% of CD3+ cells) at 6 wk postinfection. To avoid this problem, the CD8+-reconstituted RAG-/- mice were also treated with anti-CD4 Ab at the time of, and 1 wk after, adoptive transfer.

RAG-/- mice were incapable of controlling parasite growth in the site (Fig. 6GoA). At 12 wk, these mice harbored 106 parasites per ear compared with 5 x 103 in the wild-type mice. Naive CD8+ T cells failed to transfer resistance and, at 8 or 12 wk postchallenge, the number of parasites was comparable with the number estimated in the RAG-/- control. At 12 wk postinfection, RAG-/- mice reconstituted with CD4+ T were able to maintain a small number of parasites in the site (9.1 x 103/ear), while all the mice cotransferred with naive CD4+ and CD8+ T cells were able to clear the parasite from the skin (Fig. 6GoA) and the LN (data not shown). The high tissue parasite burden in the RAG-/- mice was associated with little dermal pathology (Fig. 6GoB). Surprisingly, mice reconstituted with CD8+ T cells alone developed early (3–4 wk) and severe pathology that by 10–12 wk postchallenge resulted in the complete destruction of the tissue in six of eight infected ears. Mice reconstituted with naive CD4+ T cells developed small nodules that resolved 11–12 wk postinfection. Mice reconstituted with both CD4+ and CD8+ T cells produced only a minor and transient pathology.



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  		FIGURE 6. Number of parasites per ear (A) at 8 ({diamondsuit}) or 12 (•) wk, and diameter of induration (B) following ear challenge with 1000 L. major metacyclic promastigotes in wild-type ({circ}), RAG-/- ({square}), CD8-reconstituted ({blacksquare}), CD4-reconstituted ({blacktriangleup}), or CD4 plus CD8-reconstituted (•) RAG-/- mice. Cells were purified from naive C57BL/6 mice, and were i.v. transferred at the time of ear challenge. Values represent number of parasites per individual ear, with bar indicating geometric mean, 4 mice and 4–8 ears per group; and mean induration in mm ± SD, 4–16 ears per group. Values are significantly reduced (**, p < 0.001; *, p < 0.05) compared with the parasite number in the RAG-/--unreconstituted controls. The values of only two ears are shown for the CD8-reconstituted mice at 12 wk because of the severity of the dermal erosion at this time point.

 
Immunity in RAG-/- mice reconstituted with primed CD8+ and/or CD4+ T cells

To determine whether primed CD8+ T cells could mediate protection independent of other lymphocyte populations, C57BL/6 RAG-/- mice were reconstituted with CD4+ and/or CD8+ T cells recovered from the draining LN of wild-type mice during the acute phase of infection in the skin (wk 5), and were challenged with 1000 L. major promastigotes. In each case, cell reconstitution was able to transfer resistance. At 6.5 wk, the number of parasites in the ear was reduced by 3.6 logs for CD4+-reconstituted mice and 3.7 logs for CD8+-reconstituted mice (Fig. 7GoA). One-half of the mice reconstituted with both CD4+ and CD8+ T cells did not harbor parasites in the inoculation site 6.5 wk postchallenge. RAG-/- mice reconstituted with either primed CD8+ and/or CD4+ T cells developed nodules at sites of the infection (Fig. 7GoB). Lesions were evident at 3.5 wk, progressed, and by 6.5 wk began to resolve in the mice transferred with CD4+ T cells alone or with CD8+ and CD4+ T cells. Mice transferred with primed CD8+ T cells developed a more severe lesion, but they nonetheless showed signs of healing when the experiment was terminated for parasite quantitation at 7.5 wk. In an additional experiment, it was confirmed that RAG-/- mice reconstituted with primed CD8+ T cells fully resolved their lesions by 8–10 wk.



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  		FIGURE 7. Number of parasites per ear (A) at 4 (•) or 6.5 ({blacksquare}) wk, and diameter of induration (B) following ear challenge with 1000 L. major metacyclic promastigotes in wild-type ({circ}), RAG-/- ({square}), CD8-reconstituted ({blacksquare}), CD4-reconstituted ({blacktriangleup}), or CD4 plus CD8-reconstituted (•) RAG-/- mice. Cells were purified from LN of 6-wk-infected C57BL/6 mice, and were i.v. transferred at the time of ear challenge. Values represent number of parasites per individual ear, with bar indicating geometric mean, 4 mice and 8 ears per group; and mean induration in mm ± SD, 8–16 ears per group. Values are significantly reduced (***, p < 0.001) compared with the parasite number in the RAG-/--unreconstituted controls. The experiment is representative of two separate experiments.

 
Lymphocyte recruitment and IFN-{gamma} response in reconstituted RAG-/- mice following low-dose, intradermal challenge

At 6.5 wk following transfer of primed CD8+ T cells, the draining LN of these reconstituted mice contained a population of TCR{beta}+ cells that were 91% CD8+ and <1% CD4+ (Fig. 8GoA). In the mice reconstituted with CD4+ cells, 95% of the TCR{beta} chain-positive cells were CD4+, and <2% were CD8+. The ability of the LN-reconstituted cells to produce IFN-{gamma} in response to Ag was investigated by a short restimulation in vitro using SLA, anti-CD28, and IL-2, conditions that appear to at least partially overcome the requirement for addition of infected DC to whole LN cultures. In the CD8+ T cell-reconstituted mice, 0.5% of the CD8+ cells stained for IFN-{gamma}, while 2.5% of the CD4+ T cells stained for IFN-{gamma} in the CD4+-reconstituted mice. By comparison, 1% of the CD4+ and 1.3% of the CD8+ T cells stained for IFN-{gamma} in the wild-type mice. Analysis of cells from the dermis 6.5 wk after challenge (Fig. 8GoB) revealed that both CD4+ and CD8+ cells were able to migrate to the site of parasite inoculation. A total of 4.2 x 104 CD8+ T cells per ear was recovered from the CD8+-reconstituted mice. A total of 2.5 x 105 CD4+ T cells was present in the ears of the CD4+-reconstituted mice, and 5.3% of these cells stained for IFN-{gamma} following restimulation in vitro. Mice cotransferred with CD4+ and CD8+ T cells contained both populations in the dermis (respectively, 1.3 x 105 and 2.4 x 104 cells/ear), and of these, 4.1% of the CD4+ and 8% of the CD8+ T cells were able to produce IFN-{gamma}.



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  		FIGURE 8. IFN-{gamma} production by lymphocytes present in the draining LN (A) or ears (B) of RAG-/- mice 6.5 wk following transfer of primed CD4+, CD8+ T cells, or CD4+ plus CD8+ T cells. Cells were purified from LN of 6-wk-infected C57BL/6 mice and were i.v. transferred at the time of ear challenge with 1000 L. major metacyclics. Cells were restimulated in vitro using SLA, anti-CD28, and IL-2. Dot plots of FSC/SSC and TCR{beta}+-gated cells are shown. Values in lower quadrant refer to percentage of the FSC/SSC TCR{beta}-chain-gated events positive for CD4 or CD8 markers; values in upper quadrant refer to percentage of CD4+ or CD8+ T cells positive for IFN-{gamma}, based on comparison with FL2 isotype control. The experiment is representative of two separate experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Using a challenge model that more closely reproduces the conditions of natural sand fly challenge, in which 100-1000 metacyclic promastigotes were inoculated into the ear dermis of genetically resistant C57BL/6 mice, a requirement for CD8+ T cells in the control of primary infection by L. major was revealed. C57BL/6 CD8-/- and CD8-depleted mice failed to control the growth of L. major in the skin. The control of infection in wild-type C57BL/6 mice was associated with a high number of CD8+ T cells in the dermis, a high frequency of CD8+ T cells in the draining LN able to produce IFN-{gamma} in response to parasite Ag, and the generation of primed CD8+ T cells that were able to transfer immunity to RAG-/- mice. Furthermore, reconstitution of resistance in RAG-/- mice using T cells from naive C57BL/6 mice was optimal only when both CD4+ and CD8+ T cells were transferred.

In the conventional high dose, s.c. infection model, a role for CD8+ T cells in resistance to reinfection by L. major has already been established (12). CD8+ T cells from healed mice were able to transfer Leishmania-specific delayed-type hypersensitivity to syngeneic recipients (11, 14) to express cytotoxic activity (25), and to release IFN-{gamma} in the presence of irradiated promastigotes (13, 14). The role of CD8+ T cells in the control of primary infection in genetically resistant mice remains controversial. Leishmania-reactive CD8+ T cells were found in higher numbers in resistant mice than in susceptible mice (2, 11). Depletion of CD8+ T cells increased the severity of footpad lesions in mice inoculated with a high dose of parasites; however, the mice remained able to control the infection (2). More recent studies in {beta}2-microglobulin- or CD8-deficient mice, again using a high-dose footpad challenge, showed that the mice were able to heal their footpad lesions. Taken together, these studies provide a sound basis for concluding that CD8+ T cells have little or no role in the control of primary infection by L. major (15, 16, 17).

The different outcomes obtained in these previous studies compared with the results presented in this work raise the possibility that the inoculation of an excessive dose of parasites (105-107) in an unnatural site may mask or subvert the factors responsible for the control of infection resulting from sand fly challenge. The exogenous parasite Ag provided by high-dose inocula might elicit a level of CD4+ reactivity adequate to control infection in the site, whereas intracellular infections evolving from low-dose challenge might generate a relatively poor source of Ag for class II presentation, and a requirement for both CD4+ and CD8+ T cells is revealed. Furthermore, the skin may be especially well suited for driving CD8+ T cells in response to intradermal challenge. FSDDC (26) and Langerhans cells (27) have been shown to effectively prime CD8+ T cells, and immature DC (e.g., Langerhans cells) have the capacity to induce class I-restricted CTL via presentation of Ag through an exogenous pathway (28, 29) or via presentation of peptides acquired from uptake of apoptotic cells (30).

Careful analysis of the inoculation site and draining LN in the wild-type mice provided further evidence in favor of a role for CD8+ T cells in the control of primary infection. There was a clear temporal association between reduction of the parasite load and the accumulation of CD8+ T cells in the skin able to produce IFN-{gamma}, and the presence of CD8+ T cells in draining LN expressing an activated phenotype and also able to produce IFN-{gamma}. Our results indicated that the addition of infected DC as APCs was necessary to reveal CD8+ T cell effector activity. However, the direct restimulation of the cells by anti-CD28, IL-2, and SLA could partially overcome this requirement. In cocultures involving purified populations of primed T cells, either macrophages or FSDDC were efficient APC for activation of CD4+ T cells for the release of IFN-{gamma}, while only infected or SLA-loaded DC induced the release of IFN-{gamma} by CD8+ T cells. The delayed in vivo priming of CD8+ T cells during infection may be due to the requirement for infection of DC by amastigotes released by heavily infected macrophages in the skin. These data are consistent with previous studies indicating that, in contrast to macrophages, DC can efficiently process exogenously delivered Ag for presentation by MHC class I molecules (29, 31), and that, in contrast to infected macrophages, L. major-infected DC up-regulate MHC class I and II molecules, costimulatory molecules, and IL-12 (24, 32), and effectively prime T cells in vivo (24, 33).

The experiment in RAG-/- mice reconstituted with naive lymphocytes showed that CD4+ T cells conferred a partial resistance, while CD8+ T cells transferred no protection at all. It is possible that the few contaminating CD8+ T cells contributed to the partial resistance transferred by the purified CD4+ T cells, although there was no expansion of the CD8+ cells following transfer, nor did they appear to accumulate in the site of inoculation (Fig. 8Go). Regardless, it was only when both CD4+ and CD8+ T cells were present that the parasite was cleared from the site, consistent with the fact that both populations are necessary for the expression of acquired immunity following natural challenge in intact C57BL/6 mice. The inability of naive CD8+ T cells transferred alone to develop into immune effectors suggests that CD4+ Th cells are needed to regulate their activation and/or differentiation. In contrast, primed CD8+ T cells, obtained from wild-type mice 4–5 wk following intradermal challenge, were by themselves able to transfer partial resistance to RAG-/- mice. These results formally demonstrate that effector CD8+ T cells are generated during the acute phase infection in the skin, and that they can traffic to the inoculation site and provide effector function even in the absence of CD4+ T cells. Since their capacity to produce IFN-{gamma} in response to Ag was also confirmed, this is likely to be the primary effector mechanism used by CD8+ T cells to mediate resistance. A role for CD8+ T cells producing IFN-{gamma} has been implicated in resistance to other protozoan parasites such as Toxoplasma gondii (34), Plasmodium berghei (35, 36), and Plasmodium yoelii (37). Nonetheless, a role for cytotoxic activities that might damage amastigotes (38) or release them for uptake by other cells (e.g., activated macrophages or DC) should not be ruled out. The presence of class I-restricted cytotoxic CD8+ T cells was revealed in mice that healed their primary lesion (25).

The fact that in both the naive and primed cell reconstitution experiments the most powerful resistance was observed when both populations were transferred suggests that CD4+ and CD8+ T cells provide complementary and not simply redundant effector and/or regulatory functions. For example, CD8+ T cells, via release of IFN-{gamma} or provision of CD40L, may play a regulatory role in the induction or the maintenance of a population of Th1 cells, as was recently suggested in the context of a vaccination model using LACK DNA (39). In the present studies, it is interesting that LN cells from anti-CD8-treated mice stimulated with SLA produced significantly less IFN-{gamma} than the isotype control-treated mice. Because exogenous SLA Ag added to whole LN cultures is a sufficient condition for activation of CD4+ T cells to produce IFN-{gamma} (Fig. 4Go), this result may indicate that the CD4+ T cell priming/function is impaired following CD8 depletion. Furthermore, CD8+ T cells, which have the capacity to release various chemokines (40, 41, 42), might contribute to the recruitment of CD4+ T cells to the inoculation site. It is important to note that in our preliminary studies involving adoptive transfer of positively selected CD4+ and CD8+ cells to RAG-/- mice, a minor population of CD4 cells (<1%) in the CD8 transfers resulted in a striking expansion of this population following challenge. This experience revealed a clear need to include anti-CD4 treatment in the recipient mice to maintain the purity of the CD8+ T cell reconstitution.

The delayed onset of lesion formation and slow lesion progression associated with the high dermal parasite loads in the mice with CD8+ T cell deficiencies suggests that these cells also play an important role in pathogenesis. The RAG-/- mice also had delayed lesions despite a high parasite burden in the skin. When these mice were reconstituted with CD4+ T cells, they developed a nodule at the challenge site similar to that seen in wild-type mice. These results confirm previous findings that CD4+ T cells help to mediate the pathology induced by Leishmania (19, 43). RAG-/- mice reconstituted with naive CD8+ T cells developed a severe pathology that rapidly led to the destruction of the tissue. The mechanism by which naive CD8+ T cells, when transferred alone, can aggravate pathology is not clear, but may be associated with their release of high amount of chemokines and the absence of IL-10-producing regulatory CD4+ T cells that have been shown to dampen the T cell-dependent inflammatory response to L. major in wild-type mice (44).

Our conclusions regarding the role of CD8+ T cells in pathogenesis and in the control of primary infection by L. major are consistent with clinical studies reporting high numbers of intralesional CD4+ and CD8+ T cells present during the acute stage of lesion formation and during the healing process (45, 46, 47, 48, 49, 50). The finding that CD8+ T cells are essential for development of naturally acquired immunity to primary infection has important implications regarding vaccine design. These studies provide a sound rationale for DNA vaccines, which in contrast to protein-based vaccines, have already been shown to elicit strong and durable CD8+ T cell-dependent immunity in mice (51, 52, 53).


   Acknowledgments
 
We thank Drs. Genevieve Milon and Robert Seder for critical review of this manuscript and Sandra Cooper for help with the mouse care and breeding.


   Footnotes
 
1 Address correspondence and reprint requests to Dr. David Sacks, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Building 4/126, 4 Center Drive MSC 0425, Bethesda, MD 20892-0425. E-mail address: dsacks{at}nih.gov Back

2 Abbreviations used in this paper: LN, lymph node; DC, dendritic cell; BMDC, bone marrow-derived DC; BMDM, bone marrow-derived macrophage; FSC, forward light scatter; FSDDC, fetal skin-derived DC; SLA, soluble Leishmania Ag; SSC, side light scatter. Back

Received for publication October 11, 2001. Accepted for publication February 7, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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