T Cell Vaccination in Mice with Invasive Pulmonary Aspergillosis

Mice

Inbred BALB/c (H-2^d) mice (Charles River, Calco, Italy) were used. Breeding pairs of homozygous IL-4-deficient (IL-4^−/−), IFN-γ-deficient (IFN-γ^−/−), IL-12p40-deficient (IL-12p40^−/−), and control wild-type (44, 45, 46) BALB/c mice were bred under specific pathogen-free conditions in the animal facility of the Medical School of Perugia (Perugia, Italy). Procedures involving animals and their care were conducted in conformity with national and international laws and policies. Mice of both sexes, 8–10 wk old, were used.

Micro-organism, culture conditions, and infection

The strain of A. fumigatus was obtained from a fatal case of pulmonary aspergillosis at the Infectious Diseases Institute of the University of Perugia. The micro-organism was grown on Sabouraud dextrose agar (Difco, Detroit, MI) supplemented with chloramphenicol for 4 days at room temperature. Abundant conidia were elaborated under these conditions. Conidia were harvested by washing the slant culture with 5 ml of 0.025% Tween 20 in normal saline and gently scraping the conidia from the mycelium with a plastic pipette (or by shaking it vigorously). Cell debris was allowed to settle by gravity, and the suspension was decanted into 50-ml plastic conical tubes. After extensive washing with saline, the conidia were counted and diluted to the desired concentrations. The viability of the conidia was >95%, as determined by serially diluting and plating out the inoculum on Sabouraud dextrose agar. For i.n. infection, immunosuppressed mice were lightly anesthetized with inhaled diethyl ether before instillation and for 3 consecutive days were given a daily suspension of 2 × 10⁷ conidia in 20 μl of sterile saline, slowly applied to the nostrils by micropipette with a sterile disposable tip. Animals were held in an upright position until the suspension was completely inhaled, and normal breathing resumed (27). For i.v. infection, 14 days after primary i.n. challenge, mice were injected via the lateral tail vein with 5 × 10⁶ conidia in 0.5 ml of sterile saline. Mice succumbing to fungal challenge were routinely necropsied for histopathologic confirmation of invasive aspergillosis. For histology, tissues were excised and immediately fixed in formalin. Sections (3–4 μm) of paraffin-embedded tissues were stained with periodic acid-Schiff. The Gomori methenamine silver-staining procedure was used to confirm the presence of fungal cells.

Chitin assay

For quantification of fungal growth in the organs, the chitin assay was used, as previously described (28, 29, 30). The chitin content was expressed as micrograms of glucosamine per organ. Positive control included serially diluted d(+)-glucosamine (Sigma, St. Louis, MO). The glucosamine content of lungs from uninfected mice was used as a negative control; this ranged from 0.80 to 2.25 μg of glucosamine/organ.

In vivo treatments

For immunosuppression, mice were injected i.p. with 150 mg/kg of cyclophosphamide (Sigma) 1 day before challenge. For immunization, the following Ags were used. CCFA (41) was prepared by growing the organism in a synthetic broth for 2–3 wk at 37°C. Approximately 10⁷/ml A. fumigatus conidia were inoculated into Czapek-Dox and AOAC (Association Of Analytical Chemists) broth (1/1, v/v) and incubated for 2–3 wk. The broth was separated by filtration, and the filtrate was extensively dialyzed against deionized water at 4°C. The filtrate was centrifuged, and the supernatant was freeze-dried. The protein content, reactivity with positive and negative sera, and profile in SDS-PAGE were compared with in-house standards. The freeze-dried material contained ∼40% protein by the Lowry method. The 37-kDa recombinant protein, Asp f 2, encoding an allergen of A. fumigatus was obtained as previously described (36, 43). Either type of lyophilized Ag was dissolved in distilled water and diluted to a concentration of 0.5 mg/ml sterile saline. Heat-inactivated A. fumigatus (HAF) was obtained by autoclaving A. fumigatus conidia at 121°C for 15 min. A total of 2 × 10⁷ HAF/20 μl of saline or 10 μg/20 μl of saline (according to preliminary experiments) of each Ag preparation was given i.n., 14, 7, and 3 days before the first A. fumigatus inoculation. Because immunocompetent mice quickly clear the i.n. infection and are able to survive it (30), the mice used here were immunosuppressed, unless otherwise stated, with cyclophosphamide 1 day before infection. Endotoxin was removed from all solutions with Detoxi-gel (Pierce, Rockford, IL).

Airway inflammation and collection of bronchoalveolar lavage (BAL) fluid

Airway inflammation was analyzed in the lungs as described previously (28). For enumeration of different cells, cytospin preparations of collagenase-treated lung cells were stained with May-Grünwald-Giemsa reagents (Sigma) before analysis. To improve the distinction between neutrophils and eosinophils, a modified Diff-Quik whole blood stain procedure was used. For collection of BAL fluid, lungs were lavaged thoroughly with 0.5 ml of PBS injected via the trachea. The lavage fluid was centrifuged, and the supernatant was removed and immediately stored at −80°C until analyzed for cytokine contents.

Isolation and culture of cells

The isolation of interstitial lung lymphocytes was performed as previously described (28, 29, 30). The pulmonary vasculature was perfused with 5 ml of PBS containing 100 U/ml of heparin to eliminate peripheral blood cells, and the lungs were removed, minced, and incubated for 90 min at 37°C in digestion buffer containing 0.7 μg/ml collagenase (Sigma) and 30 μg/ml type IV bovine pancreatic DNase I (Sigma). Large particulate matter was removed by passing the cell suspension through a small, loose, nylon wool plug, and lymphocytes were enriched over a single-step Ficoll gradient. Cells were resuspended in RPMI containing 10% FCS, 2-ME (50 mM), sodium pyruvate (1 mM), HEPES (10 mM), and gentamicin (50 μg/ml; complete medium) and were plated at a concentration of 2 × 10⁵/200 μl in 96-well microtiter plates (Falcon Labware, Becton Dickinson, Franklin Lakes, NJ) coated with 12 μg/ml hamster anti-mouse CD3 mAb (clone 32C11, PharMingen, San Diego, CA). After 48-h culture, supernatants were harvested, and cytokine production was determined by specific ELISA. CD4⁺ T cells (>95% pure on FACS analysis) were purified from lungs or spleens using anti-mouse CD4-conjugated magnetic MicroBeads (Miltenyi Biotech, Bergisch Gladbach, Germany). Briefly, 1 × 10⁷ total cells were incubated with 10 μl of MACS, magnetic cell sorting of CD4⁺ MicroBeads for 15 min at 6°C, washed, and magnetically separated with a positive selection column, according to the manufacturer’s instructions. Dendritic cells (DC) were separated from lungs and spleens using N-418-conjugated MicroBeads (Miltenyi Biotech) followed by magnetic separation according to the manufacturer’s instructions. On FACS analysis, the purified populations reacted positively with mAb to FcRII, C3biR, and mAb NLDC-145 and were negative upon staining with mAb F4/80 as described previously (47, 48).

Adoptive transfer of T cells

An adoptive transfer model known to modulate airway immune reactivity in a mouse model of asthma was adopted (49). Mice were injected with CCFA or Asp f 2 Ags or with vehicle alone (hereafter referred to as untreated) as described above before receiving the i.n. challenge with Aspergillus conidia (2 × 10⁷ conidia/injection, three times). Treatment with cyclophosphamide was purposely omitted so as not to compromise splenic cell recovery and reactivity. For adoptive transfer, CD4⁺ T cells were purified from spleens 14 days after the i.n. challenge and restimulated in vitro with DC from the same mice and the relevant Ag for 3 days before cell and supernatant collection. Flow cytometric analysis revealed that cultured lymphocytes from CCFA-treated mice consisted of CD3⁺ cells (95% in both populations from CCFA-treated and mock-treated mice), CD4⁺ cells (87 and 90%), CD8⁺ cells (9 and 7%), CD44^high (99 and 43%), and Mel-14^low (70 and 23%). Five × 10⁵ cells were injected i.v. to naive recipients 1 day before the i.v. challenge with 5 × 10⁶ Aspergillus conidia.

Cell proliferation assay

Purified lung CD4⁺ T cells (5 × 10⁵) were cultured with 5 × 10⁴ purified lung DC in 200 μl of complete medium, in flat-bottom 96-well microtiter plates (Falcon) in the presence of 1 μg/ml CCFA or Asp f 2 or 1 μg/ml Con A for 4 days at 37°C in 5% CO₂ (according to preliminary experiments indicating the doses at which optimal proliferation is obtained). Eight hours before harvesting, cells were pulsed with 0.5 μCi of [³H]thymidine/well. Incorporation into cellular DNA was measured by liquid scintillation counting. The results are expressed as the mean counts per minute ± SE.

Cytokine and IgE assays

The levels of IL-12p70 and IL-10 in BAL fluids and of IFN-γ, IL-2, and IL-4 in culture supernatants of activated cells were determined by cytokine-specific ELISA, using pairs of anti-cytokine mAbs as described previously (28, 29, 30). The Ab pairs used were as follows, listed by capture/biotinylated detection: IFN-γ, R4-6A2/XMG1.2; IL-2, JES6-1A12/JES6-5H4; IL-4, BVD4-1D11/BVD6-24G2; and IL-10, JES5-2A5/SXC-1 (PharMingen). IL-12p70 was determined using an ELISA kit (R&D Systems, Space Import-Export, Milan, Italy). Cytokine titers were calculated by reference to standard curves constructed with known amounts of recombinant cytokines (from PharMingen or, for IL-12, R&D Systems). A micro-ELISA procedure was used to quantitate total IgE in the BAL fluid as described previously (29, 46).

RNA preparation and RT-PCR

Total lung cells were subjected to RNA extraction by the guanidium thiocyanate-phenol-chloroform procedure, as previously described (28, 29, 30). Briefly, 5 μg of total RNA was reverse transcribed into cDNA using Moloney murine leukemia virus RT (Life Technologies, Gaithersburg, MD). The cDNA was then amplified using specific primers for murine IFN-γ, IL-4, IL-10, IL-12 receptors β1 and β2 (IL-12Rβ1 and IL-12Rβ2), and hypoxanthine-guanine phosphoribosyl transferase (HPRT) as described previously (28). Amplifications were performed in 2 mM MgCl₂, 50 mM KCl, 10 mM Tris-HCl, (pH 8.3), 0.2 mM of each deoxynucleotide triphosphate, 1 μM of each primer, and 2.5 U of AmpliTaq polymerase (Perkin-Elmer/Cetus, Norwalk, CT). The cDNA was amplified in an automated thermal cycler (Perkin-Elmer/Cetus) as described previously (28). Amplification was stopped at 35 cycles. The HPRT primers were used as a control for both RT and the PCR reaction and also for comparing the amounts of products from samples obtained with the same primer. The PCR fragments were analyzed by 1.5% agarose gel electrophoresis, stained with 0.5 mg/ml ethidium bromide, and visualized using a UV transilluminator.

Statistical analysis

Survival data were analyzed using the Mann-Whitney U test; significance was defined as p ≤ 0.05. Student’s t test was used to determine the statistical significance of values (mean ± SE) between experimental groups. In vivo groups consisted of four to six animals. The data reported were pooled from at least three experiments.

Effect of administration of Aspergillus or different Aspergillus Ags on the course of IPA

Multiple i.n. inoculations of A. fumigatus conidia in immunocompetent DBA/2 mice caused a transient, self-limiting infection that conferred immune resistance to subsequent infection (28). Here we show that a similar result was obtained in BALB/c mice, in that mice surviving an i.n. exposure to Aspergillus conidia also showed increased resistance to local or systemic subsequent infection, as indicated by the increased survival and decreased fungal growth in organs (Table I⇓). Interestingly, no protection was observed following a primary intragastric Aspergillus infection (data not shown). On assessing the capacity of inactivated Aspergillus conidia or different Aspergillus Ags to induce protection to subsequent infection, we found that treatment with HAF or Asp f 2 did not protect mice against the i.n. or i.v. infection. Coadministration of IL-12 did not induce protection (data not shown). In contrast, immunization with CCFA induced significant protection against both types of infection, as judged by marked reduction of the fungal growth in the organs and the ability of the majority (>80%) of mice to survive an otherwise lethal intranasal infection or to partially resist the i.v. infection. Remarkably, CCFA-induced protection was also observed in DBA/2 mice (data not shown), which are more susceptible than BALB/c mice to pulmonary and invasive aspergillosis (15, 28).

CCFA-induced protection correlated with the local recruitment of lymphocytes and macrophages more than neutrophils, as indicated by cytospin preparations of collagenase-treated lungs and lung histology. We have shown that leukocyte recruitment into the lungs of infected and immunosuppressed mice occurred despite a profound leukopenia (28). The percentages of lymphocytes (30%) and macrophages (36%) recruited at the site of infection were significantly higher in CCFA-treated mice than in Asp f 2-treated mice (17 and 22%, respectively) or untreated mice (16 and 20%, respectively). In addition, the percentages of neutrophils (33%) and eosinophils (1%) were significantly lower in CCFA-treated mice than in Asp f 2-treated (61 and 10%, respectively) or untreated mice (56 and 8%, respectively). Lung sections from CCFA- or Asp f 2-treated or untreated mice revealed patterns of lesions similar to those observed in invasive and noninvasive pulmonary aspergillosis (28, 29, 30). Resistant, CCFA-treated mice revealed the presence of few infiltrates, consisting of rare, small hyphae with a moderate infiltration of predominant inflammatory mononuclear cells in the peribronchial region (Fig. 1⇓A). In contrast, lung sections from Asp f 2-treated mice (Fig. 1⇓B) revealed patterns of lesions similar to those observed in untreated mice (Fig. 1⇓C), characterized by signs of bronchial wall damage, peribronchial necrosis, and the presence of numerous infiltrating (predominantly polymorphonuclear) cells.

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FIGURE 1.

Lung histology of mice with invasive aspergillosis upon treatment with different Aspergillus Ags. Periodic acid-Schiff-stained sections were prepared from lungs of BALB/c mice treated with Aspergillus crude culture filtrate Ags (A) or the recombinant protein Asp f 2 (B) or were untreated (C), and infected i.n. with Aspergillus conidia after being immunosuppressed with cyclophosphamide 1 day before the infection. Lungs were taken 5 days after infection. A, Note the presence of few infiltrates, consisting of rare, small hyphae with moderate infiltration of predominant inflammatory mononuclear cells in the peribronchial region (inset of A). In contrast, the sections from the lungs of Asp f 2-treated mice (B) revealed patterns of lesions similar to those observed in untreated mice (C), characterized by signs of bronchial wall damage, peribronchial necrosis, and the presence of predominant infiltrating neutrophils (insets of B and C). Bar, 400 μm; 100 μm in the inset.

Proliferative responses to Aspergillus Ags

To evaluate whether the activation of local, specific T lymphocytes to Aspergillus Ags had occurred in infected mice pretreated with the different Ags, lung lymphocytes or purified CD4⁺ T cells were assessed for proliferation in vitro. Cells from untreated mice, CCFA-treated mice, or Asp f 2-treated mice were cultured in vitro with the relevant Ags in the presence of lung DC from the same mice (in the case of CD4⁺ T cells). The results (Fig. 2⇓A) indicate that lung cells from untreated or CCFA-treated mice proliferated in response to Con A and to some extent in response to the CCFA or Asp f 2 Ags. However, in untreated mice CD4⁺ T cells only proliferated in response to Con A (Fig. 2⇓B), while in mice immunized with CCFA, CD4⁺ T cells strongly reacted to the CCFA Ags, but not to the Asp f 2 Ag (Fig. 2⇓B). Treatment with Asp f 2 did not induce Ag-specific proliferation (Fig. 2⇓). Thus, the proliferative response to Asp f 2 Ag observed in mice untreated or treated with CCFA may be due to cells other than CD4⁺ lymphocytes.

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FIGURE 2.

In vitro proliferative response of unfractionated (A) or CD4⁺ (B) lung cells from mice with invasive aspergillosis, either untreated or treated with Aspergillus CCFA or the recombinant protein Asp f 2. Cells were unstimulated (□) or were stimulated with Con A (▥), Aspergillus CCFA (▪), or the recombinant protein, Asp f 2 (▧), with (B) or without (A) purified lung DC (see Materials and Methods for details), for 4 days at 37°C in 5% CO_2. Eight hours before harvesting, cells were pulsed with 0.5 μCi of [³H]thymidine/well. Results are expressed as the mean counts per minute ± SE of triplicate samples. ∗, p < 0.05, mitogen- or Ag-stimulated vs unstimulated.

Production of cytokines and IgE in mice treated with different Aspergillus Ags

To look for patterns of cytokine and Ab production in response to the different Aspergillus Ags, the levels of IL-12p70, IL-10, and IgE were determined in BAL fluids and those of IFN-γ, IL-2, and IL-4 were determined in culture supernatants of anti-CD3-stimulated lung lymphocytes soon after infection. The results (Fig. 3⇓) show that 3 days after infection, the production of IL-12p70 was significantly increased, while that of IL-10 was significantly decreased in BAL from CCFA-treated mice with respect to untreated or Asp f 2-treated animals. The production of IFN-γ and IL-2 was also significantly increased in CCFA-treated mice 7 days after infection at the time when the levels of IgE in BAL were significantly lower than those found in untreated or Asp f 2-treated mice (Fig. 3⇓). The production of IL-4 was significantly increased upon Asp f 2 treatment. Studies performed on IL-12-, IFN-γ-, or IL-4-deficient mice revealed that the immunization capacity of CCFA was lost in the absence of IL-12 and IFN-γ and was potentiated in the absence of IL-4. Indeed, the fungal growth in the lungs was not inhibited in the absence of IL-12 and IFN-γ, but was greatly reduced in the absence of IL-4 (Table II⇓).

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FIGURE 3.

Cytokine and IgE contents in mice with invasive pulmonary aspergillosis, either untreated (□) or treated with Aspergillus CCFA (▪) or the recombinant protein Asp f 2 (▧). IL-12p70 and IL-10 were determined in the BAL fluids 3 days after infection, and IgE was determined 7 days after infection. IFN-γ, IL-2, and IL-4 were determined in culture supernatant of anti-CD3-activated lung cells 7 days after infection. Culture supernatants of lung cells were collected after 48-h incubation at 37°C in 5% CO₂. Cytokine and IgE levels were determined by specific ELISA. Cytokine contents in cultures from uninfected and untreated mice were below the detection limit of the assay (indicated by < on the y-axis). ∗, p < 0.05, CCFA-treated or Asp f 2-treated vs untreated.

Adoptive transfer of splenic CD4⁺ T cells

To assess whether the induction of Ag-specific CD4⁺ T lymphocytes, producing IFN-γ and IL-2, is correlated with protection against IPA, splenic CD4⁺ T cells from untreated, CCFA-treated, or Asp f 2-treated mice were cultured in vitro with the relevant Ag and DC, and adoptively transferred into naive BALB/c mice 1 day before i.v. infection with A. fumigatus. The results (Table III⇓) indicate that adoptive transfer of CD4⁺ T cells from CCFA-treated mice significantly prolonged survival after the infection, while adoptive transfer of cells from Asp f 2-treated or untreated donors did not. On looking at the pattern of cytokine and cytokine receptor gene expression by adoptively transferred cells, we found that the messages for IFN-γ and IL-12Rβ2 were both expressed in cells from CCFA-treated mice, while those for IL-4 and IL-10 were almost undetectable. In contrast, the IL-12Rβ2 message was undetectable in cells from untreated or Asp f 2-treated mice, while those of IL-4 and IL-10 were present (Fig. 4⇓). Because the expression of the IL-12Rβ2 positively correlated with the induction of Th1 lung lymphocytes in mice with aspergillosis (30), these results indicate that the transferred cells from CCFA-treated mice were of the Th1 phenotype.

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FIGURE 4.

Cytokine and cytokine receptor gene expression in CD4⁺ T cells from spleens of mice either untreated (lane 1) or treated with Aspergillus CCFA (lane 2) or the recombinant protein Asp f 2 (lane 3) before i.n. infection with Aspergillus conidia. CD4⁺ T cells were purified 14 days after i.n. infection and were restimulated in vitro with the relevant Ags and purified DC for 3 days before cell collection and adoptive transfer into naive recipients. Cytokine and cytokine receptor gene expression were assessed by RT-PCR. C, specific positive controls for HPRT or cytokines or IL-12R β-chains; N, no DNA added to amplification mix during PCR.