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Murine CD4+ T Lymphocyte Subsets and Host Defense against Pneumocystis carinii

  1. Judd E. Shellito1,
  2. Chandra Tate1,
  3. Sanbao Ruan1 and
  4. Jay Rolls1,2
  1. 1Section of Pulmonary and Critical Care Medicine, Department of Medicine, New Orleans, Louisiana
  2. 2Gene Therapy Program, Louisiana State University Health Sciences Center, New Orleans, Louisiana
  1. Reprints or correspondence: Dr. Judd E. Shellito, Section of Pulmonary and Critical Care Medicine, Louisiana Health Sciences Center, 1901 Perdido St., Ste. 3205, New Orleans, LA 70112 (jshell{at}lsumc.edu).

  1. Presented in part: annual meeting of the American Thoracic Society, San Diego, May 1999 (abstract A228).

 
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Abstract

The recruitment of specific subsets of CD4+ T lymphocytes to the lungs in response to Pneumocystis carinii was investigated. For mice inoculated with P. carinii, an ELISPOT assay was used to calculate the numbers of lymph node and lung tissue CD4+ cells that secreted interferon (IFN)-γ (Th1 cytokine) and interleukin (IL)-4 (Th2 cytokine) after concanavalin A stimulation. An ELISA was used to assay culture supernatants for cytokine concentrations. Precursor frequency of both IFN-γ- and IL-4-secreting cells was increased in lymph nodes at 1 week, whereas increases in Thl and Th2 cells in lung tissue were delayed 3 weeks before declining. The frequency of IL-4-secreting cells always was greater than the frequency of IFN-γ secreting cells. These results demonstrate an early T lymphocyte response in draining lymph nodes, followed by later recruitment of Thl and Th2 lymphocytes into lung tissue. The overall CD4+ T cell response to P. carinii involves both Thl and Th2 subsets, but the response is Th2 dominant in both lymph node and lung tissue.

Pulmonary infection with Pneumocystis carinii is the most common severe opportunistic infection that complicates human immunodeficiency virus (HIV) infection [1, 2]. Host defense mechanisms against P. carinii are poorly understood. Human infection with P. carinii is associated more with defects in cell-mediated immunity, such as HIV infection, than with neutrophil dysfunction. As a result, P. carinii infections have become a particular clinical problem associated with HIV infection. The risk of an HIV-infected subject's acquiring P. carinii pneumonia shows a linear and inverse correlation with the number of circulating CD4+ lymphocytes [3]. The importance of the CD4+ T lymphocyte in host defense against P. carinii is further supported by work from our laboratory that shows that normal mice inoculated with P. carinii can resolve the infection without treatment, while mice that are depleted of CD4+ T lymphocytes with a monoclonal antibody develop progressive P. carinii pneumonia [4]. When administration of the CD4+ antibody is stopped and CD4+ lymphocytes are restored in lymphoid tissue, P. carinii organisms are cleared from lung tissue, and infection resolves [5]. Collectively, our study and the studies of others [6–8] support a key role for the CD4+ helper T lymphocyte in host defense against P. carinii.

All CD4+ T lymphocytes do not have identical functional capacities. Cloned lines of murine CD4+ T lymphocytes can be divided into functionally discrete subsets, designated Thl and Th2, on the basis of the types of cytokines they produce in vitro [9, 10]. Thl cells produce interleukin (IL)-2, interferon (IFN)-γ, and tumor necrosis factor (TNF)-β, whereas Th2 cells produce IL-4, -5, -6, -10, and-13 [11]. The cytokines IL-3, TNF-α, and granulocyte-macrophage colony-stimulating factor are produced by both subsets. Thl responses, through the elaboration of IFN-γ and macrophage activation, favor cell-mediated immunity, whereas Th2 responses, through the elaboration of IL-4 and other cytokines, favor humoral immune responses [12, 13]. Emerging evidence supports the existence of T lymphocyte subsets in humans, as well as in mice [14, 15].

The role of T helper subsets in different disease processes is a subject of intense research, but it is clear that these subsets are important in host defense against infection. This has been best demonstrated in murine infection with Leishmania major. Mouse strains that produce a Thl response clear the infection, whereas strains with a Th2 response show disease progression [16, 17]. Similar information has been reported in regard to animal infection with Mycobacterium tuberculosis [18] and with cryptococcosis [19] and to human infection with M. leprae [20, 21]. The role of T lymphocyte subsets in host defense against infection with P. carinii is unknown.

In our study, we evaluated CD4+ T lymphocytes in the draining of lymph nodes and lung tissue of normal mice inoculated with P. carinii organisms at serial intervals, from 1–4 weeks. Other experiments have demonstrated that normal mice clear inoculated organisms within this time period and do not become chronically infected [4]. Recovered cells were assayed for the release of cytokines that correspond to the Th1 or Th2 phenotype after in vitro stimulation with the mitogen concan-avalin A (ConA).

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Materials and Methods

Animals

Specific pathogen-free female BALB/c mice were purchased at age 8–9 weeks (weight, 25 g) from Hilltop Laboratories (Scottsdale, PA). The animals were housed in filter-topped cages in an isolation room at the animal care facility. Access to the room is limited to certain laboratory and animal care personnel, and gown and gloves are required to enter the room. All caging procedures and surgical manipulations were done under a laminar flow hood. Mice were fed autoclaved standard chow and water ad libitum and were kept in the facility for at least 2 days before any treatment was begun. To exclude viral infection, sentinel DBA mice were housed with bedding used by P. carinii-infected mice. Plasma titers against a battery of murine viruses and tissue cultures were done quarterly in the sentinel mice.

P. carinii inoculation. P. carinii for inoculation was prepared as described elsewhere [5], by using lung homogenates from CB17 scidlscid mice (Taconic Farms, Germantown, NY) that were chronically infected with P. carinii (initial infected animals used to establish the colony were purchased from Fox Chase Cancer Center, Philadelphia). In brief, scid mice with chronic P. carinii infection were injected intraperitoneally (ip) with a lethal dose of pentobarbital (200 mg/kg) and were exsanguinated by aortic transection. The lungs were removed aseptically and were frozen for 30 min in 1 mL of PBS at −70°C. Frozen lungs were homogenized (Model 80 Stomacher; Tekmar Instruments, Cincinnati) in 10 mL of PBS, were forced through a sterile 70-mm nylon strainer (Falcon 2350; Becton Dickinson, Lincoln Park, NJ), and were pelleted at 500 g for 10 min at 40°C. The pellet was resuspended in PBS, and a 1 : 4 dilution was stained with modified Giemsa stain (Diff-Quik; Baxter, McGraw Park, IL). The number of P. carinii cysts was quantified microscopically, and the inoculum concentration was adjusted to 2 × 106 cyst/mL. Gram's stain was used on the inoculum to exclude overt contamination with bacteria. Random endotoxin levels in the inoculum were <0.01 ng/mL, by the limulus amoebocyte lysate assay.

Recipient mice were anesthetized ip with pentobarbital (75 mg/kg) and then inoculated. After the anterior side of the neck was swabbed with isopropyl alcohol, a midline skin incision was made to expose the trachea. Next, an 18-gauge blunt-ended needle was introduced into the trachea through the mouth, and 0.1 mL of P. carinii inoculum (2 × 105 P. carinii cysts) was injected through a 22-gauge inner needle into the lungs, followed by 0.5 mL of air. This dose was chosen because it causes a reproducible P. carinii infection 4 weeks after inoculation into mice deficient in CD4+ T lymphocytes, whereas normal mice clear the infection during this time period [5]. Control mice were inoculated with an equal volume of lung tissue from uninfected BALB/c mice. This control inoculum was prepared in an identical fashion to that from P. carinii-iniected mice and was matched to protein content (Pierce Chemical, Rockford, IL). The incision was closed with a wound clip, and the mice were allowed to recover. All needles and instruments were baked at 170°C to prevent contamination with endotoxin. PE-10 tubing was sterilized with isopropyl alcohol.

Isolation of CD4+ T lymphocytes from hilar and paratracheal lymph nodes

At serial times after intratracheal inoculation, animals were given a lethal dose of pentobarbital (200 mg/kg) and were exsanguinated by aortic transection. Hilar lymph nodes and paratracheal (mediastinal) lymph nodes then were resected under sterile conditions. This method has been used by others to study the draining of lymph node cells from mice challenged with antigen [22, 23]. Resected lymph nodes were passed through a sterile mesh screen into culture medium and were adjusted for cell number with a hemocytometer. By using this technique, ∼12–15 × 106 cells were recovered from a mouse inoculated with P. carinii. These cells were >90% lymphocytes, according to modified Giemsa staining.

CD4+ T lymphocytes were purified from lymph node cells, by using L3T4 antibody-coated magnetic beads (Dynal, Lake Success, NY). Magnetic beads were added to cells in a 15-mL polypropylene tube (107 beads/2.5 × 106 lymph node cells), were vortexed gently, and were incubated at room temperature for 20 min on a tilting platform. The cells and beads then were placed in a magnet column for 2 min before the supernatant was aspirated. The centrifuge tube then was removed from the magnet, and bound cells were resuspended in buffer. This procedure was repeated 3 times to fully recover CD4+ cells from the cell preparation. Magnetically separated cells were detached from the magnetic beads by incubation with a polyclonal anti-Fab antibody (DETACHaBEAD; Dynal) specific for the antibodies on the magnetic beads. Cells bound to beads were suspended in culture medium at 107 cells/mL. The anti-Fab antibody then was added at 10 μL/100 μL of cell suspension, was vortexed gently, and was incubated for 45 min at room temperature on a tilting platform. The beads then were removed from the cell suspension, by using the magnet column. Detached CD4+ T lymphocytes were washed 2 times to remove the anti-Fab antibody and were resuspended in culture medium. When used by us, this technique isolated CD4+ T lymphocytes to 86.7% + 2.2% purity, by flow cytometry.

Isolation of CD4+ T lymphocytes from lung tissue

Interstitial lymphocytes were isolated from enzyme-digested lung tissue [24–26]. In brief, lungs were excised and dissected free of blood vessels and large airways. The lungs then were minced with scissors in RPMI that contained 150 U/mL collagenase (Worthington Biochemical, Freehold, NJ) and 50 U/mL DNAse (Sigma, St Louis) at 10 mL enzyme mixture for each 0.6 g of lung tissue. Lung tissue was digested for 90 min at 37° C. Digested lung then was filtered through a mesh screen, to remove tissue fragments. Recovered cells were washed, counted in a hemocytometer, and purified for CD4+ T lymphocytes, by using magnetic beads as described above. It should be noted that murine CD4 is exclusive to T lymphocytes and is not expressed on macrophages as it is in humans [27].

ELISPOT assay for IFN-γ- and IL-4-secreting cells

The ELISPOT assay for frequency of cells that secrete IFN-γ and IL-4 was applied to purified CD4+ T lymphocytes from lymph nodes and lung tissue in a modification of the method described by Czerkinsky et al. [28]. In brief, selected wells in a 96-well cellulose membrane plate (Millipore, Bedford, MA) first were coated with anti-IFN-γ antibody (6 μg/mL R4-6A2; PharMingen, San Diego) or anti-IL-4 antibody (10 μg/mL 1D11; Endogen, Woburn, MA) for 24 h at 4°C. The plates then were blocked by incubation with 5% bovine serum albumin for 30 min at 37°C. The CD4+ T lymphocytes were added to washed wells in 100 μL of culture medium (RPMI supplemented with 10% fetal bovine serum [FBS], 50 μM 2-mercaptoethanol, 0.1 mg/mL gentamicin, 100 U/mL penicillin, 100 μg/mL streptomycin, and 2 mM L-glutamine). The concentration of cells added to each well is critical to allow for readable numbers of spots. For lymph node CD4+ cells, we added 105 cells for the detection of IFN-γ and 0.5 × 105 cells for the detection of IL-4. For lung tissue CD4+ cells, we added 0.5 × 105 cells for the detection of IFN-γ and 0.25 × 105 cells for the detection of IL-4.

The cells were incubated on the antibody-coated plates at 37°C in 5% CO2 in air for 22 h. Duplicate wells were cultured with (5 mg/μL) and without added ConA (Sigma). The plates were washed 3 times with PBS and then 3 times with PBS-0.05% Tween to remove bound cells. Biotinylated detecting antibody then was added to appropriate wells at 4 μg/mL for biotinylated anti-IFN-γ (XMG1.2; PharMingen) and at 3 μg/mL for biotinylated anti-IL-4 (24G2; Endogen).

Plates were incubated at 4°C for 18 h. Wells were washed 4 times with PBS-Tween, and 100 μL of streptavidin-conjugatedperoxidase (1 : 400 dilution of 1 mg in 1% PBS-Tween; Sigma) was added to each well. The plates were incubated with peroxidase for 2 h at room temperature in the dark and then were washed 3 times with PBS-Tween. Substrate (AEC; Sigma) then was prepared according to the manufacturer's directions and was added at 200 μL/well. The plates were incubated for 15–30 min to develop spots and then were washed with water. Plates were shaken free of water and were air dried before the spots were counted under a dissecting microscope. Spots were counted in duplicate wells, and data were expressed as the number of IFN-γ or IL-4 spots/105 CD4+ T lymphocytes.

Assay of cytokine release from CD4+ T lymphocytes after in vitro culture

Concentrations of cytokine proteins (IFN-γ and IL-4) were assayed in supernatants of lymphocyte cultures by ELISA. Purified CD4+ T lymphocytes from lymph nodes and lung digest were plated in 96-well microtiter plates at 106 cells/well in RPMI that contained 5% FBS and 1 mg/μL ConA. After 24 h of culture at 37°C in 5% CO2 in air, the plates were centrifuged, and culture supernatants were aspirated. Cytokine concentrations in culture supernatants were assayed by sandwich ELISA kits (murine IFN-γ; Genzyme, Cambridge, MA; and murine IL-4; R&D Systems, Minneapolis).

Antigen-induced proliferation of CD4+ T lymphocytes

P. carinii organisms were isolated from the lungs of chronically infected scid mice, by using the method described by Kaneshiro et al. [29]. Infected lung tissue was minced with scissors and was resuspended in NaCa HEPES buffer that contained 0.5% glutathione. This was filtered through a 70-mm mesh screen, and the filtrate was centrifuged at 925 g for 10 min. Host cells were lysed with 0.85 M NH4CL for 15 min, followed by another centrifugation at 925 g. The pellet then was resuspended in NaCa HEPES buffer and was centrifuged at 60 g. The resulting supernatant was centrifuged at 925 g, was resuspended, and was subjected to another round of slow-speed, followed by high-speed, centrifugation. Last, the resultant pellet was gravity filtered through an 8-μm filter.

Microscopic examination of Giemsa-stained organisms purified in this manner showed trophic forms almost exclusively, with lesser numbers of cyst forms. There were no visible intact host cells, although an occasional cell nucleus was seen. Purified organisms were examined by Gram's stain and culture to check for bacterial contamination and then were suspended in PBS at 1 mg/mL protein (Pierce). Aliquots of purified P. carinii were frozen at −100°C and were used within 6 months of preparation.

To assay in vitro proliferation of CD4+ T lymphocytes, purified CD4+ T cells from lymph node and lung were cultured with graded concentrations of purified P. carinii organisms and irradiated spleen cells from normal mice. Control cultures contained equivalent concentrations of ovalbumin (OVA). In brief, CD4+ T lymphocytes purified from lungs and lymph nodes as described above were added in culture medium (RPMI supplemented with 10% FBS, 50 μM 2-mercaptoethanol, 10 mM HEPES buffer, 0.1 mg/mL gentamicin, 200 U/mL penicillin, 100 μg/mL streptomycin, and 2 mM L-glutamine) to 96-well microtiter plates at 2 × 105 cells/well. To provide accessory cell activity, 5 × 105 γ-irradiated spleen cells from a normal mouse were added to each well. Cells were cultured for 3–5 days at 37°C in 5% CO2 in air. At the termination of the culture, cells were labeled with bromodeoxyuridine (BrdU; Boehringer Mannheim, Mannheim, Germany) for 2 h, and BrdU uptake was measured in an ELISA plate reader.

Statistical analysis

Data were analyzed by using a Student's t test or analysis of variance, followed by the Newman-Keuls test. Statistical significance was set at P ⩽.05. All experiments were repeated more than once. The data represented in the results section are mean ± SD of all results.

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Results

Recovery of CD4+ T lymphocytes from lymph node and lung tissue

CD4+ T lymphocytes were recovered from lymph node and lung tissue at serial intervals (1–4 weeks) after inoculation with P. carinii. Normal mice will clear inoculated organisms from lung tissue during this time [5]. To control for nonspecific inflammation induced by inoculation of lung tissue, control mice received an equivalent amount of lung tissue from uninfected mice. We found that numbers of CD4+ T lymphocytes were increased significantly in regional lymph node tissue, compared with control lung tissue, at 1, 2, and 3 weeks after inoculation with P. carinii (figure 1A). CD4+ T lymphocytes in lymph nodes declined to control levels by 4 weeks. In contrast, numbers of CD4+ T lymphocytes in lung tissue after inoculation with P. carinii were increased only at 1 week and then remained at control levels for 2–4 weeks (figure 1B).

Figure 1
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Figure 1

No. of CD4+ T lymphocytes isolated from lymph node (A) and enzyme-digested lung tissue (B) of mice at serial intervals after inoculation with Pneumocystis carinii or control lung tissue. n = 6 for each data point. Results are expressed as CD4+ cell number X 106/mouse. *P⩽ .05, compared with control lung tissue.

ELISPOT profiles for lymph node CD4+ T lymphocytes

Precursor frequencies of IFN-γ- and IL-4-producing cells were increased significantly in lymph nodes early (1–2 weeks) after P. carinii inoculation and then declined to control frequencies (figure 2). Lymph node CD4+ T lymphocyte responses were Th2 dominant at all time intervals, in that IL-4 precursor frequency (figure 2B) was greater than IFN-γ precursor frequency (figure 2A). Note the difference in scale for the y axes in figure 2. For comparison purposes, CD4+ T lymphocytes from lymph nodes of uninoculated control mice contained 4 IFN-γ spots/105 cells and 10 IL-4 spots/105 cells.

Figure 2
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Figure 2

Precursor frequency of CD4+ T lymphocytes that secreted interferon (IFN)-γ (A) and interleukin (IL)-4 (B) isolated from lymph nodes of mice at serial intervals after inoculation with Pneumocystis carinii or control lung tissue, n = 6 for each data point. Results are expressed as the number of spots for each cytokine/105 CD4+ T lymphocytes. *P ⩾ .01, compared with control lung tissue.

ELISPOT profiles for lung tissue CD4+ T lymphocytes

Precursor frequencies of IFN-γ- and IL-4-producing CD4+ T lymphocytes derived from lung tissue were increased at time intervals later than were those for lymph nodes (figure 3). Maximal precursor frequencies for both IFN-γ- and IL-4-producing cells were observed 3 weeks after inoculation of P. carinii. As in lymph nodes, CD4+ T lymphocyte responses were Th2 dominant at all time intervals. Again, note the difference in scale for the y axes in figure 3. For comparison purposes, CD4+ T lymphocytes from lung tissue of uninoculated control mice contained 8.5 IFN-γ spots/105 cells and 32 IL-4 spots/105 cells.

Figure 3
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Figure 3

Precursor frequency of CD4+T lymphocytes that secreted interferon (IFN)-γ (A) and interleukin (IL)-4 (B) isolated from lung tissue of mice at serial intervals after inoculation with Pneumocystis carinii or control lung tissue, n = 6 for each data point. Results are expressed as the number of spots for each cytokine/105 CD4+ T lymphocytes. *P ⩽ .01, compared with control lung tissue.

Cytokine release by CD4+ T lymphocytes in vitro

Culture supernatants of CD4+ T lymphocytes from lymph nodes of mice inoculated with P. carinii had increased concentrations of IL-4, compared with the concentrations for control mice at both 1 and 3 weeks (table 1). Compared with lung tissue from control animals, lymph node CD4+ T lymphocytes from animals inoculated with P. carinii had no change in the capacity to release IFN-γ. In contrast, CD4+ T lymphocytes from lung tissue had no change in the capacity to release IL-4 in vitro; however, they did have an increased release of IFN-γ in vitro for lung CD4+ T lymphocytes recovered 3 weeks after P. carinii inoculation.

Table 1
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Table 1

In vitro cytokine release (in pg/mL) by concanavalin A-stimulated CD4+ T lymphocytes from lymph nodes and lung tissue of Pneumocystis carinii-infeted and control mice.

Antigen-induced proliferation of CD4+ T lymphocytes in vitro

To assess the capacity of lung T lymphocytes to respond to specific stimulation with P. carinii, mice were killed 3 weeks after intratracheal inoculation with P. carinii and were cultured in vitro with P. carinii antigen and with irradiated accessory cells. Control cultures were stimulated with equivalent amounts of OVA. Lung lymphocytes from mice inoculated with P. carinii were stimulated to proliferate by P. carinii antigen in a dose-dependent manner (0.35 ± 0.04 absorbance units [AU] for 1 μg/mL P. carinii and 0.4 ± 0.15 AU for 10 μg/mL P. carinii; P < .05, compared with OVA). OVA added to the cultures did not stimulate lymphocyte proliferation above background levels without added protein (0.28 ± 0.02 AU for 10 μg/mL OVA). Similar but lesser amounts of BrdU incorporation were observed after 3 days of in vitro culture (data not shown). CD4+ T lymphocytes from control mice did not proliferate in vitro in response to added P. carinii antigen (0.27 ± 0.02 AU with 100 μg/mL P. carinii protein after 5 days of culture).

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Discussion

In these studies, we characterized CD4+ T lymphocyte responses to P. carinii in lymph nodes and in lung tissue according to numbers of CD4+ T lymphocytes, precursor frequencies of IFN-γ-producing (Th1 surrogate) and IL-4-producing (Th2 surrogate) cells, capacity to release IFN-γ or IL-4 in vitro, and capacity to proliferate in response to P. carinii antigens. Our data demonstrate an early increase in numbers of CD4+ T lymphocytes in draining lymph nodes associated with an increased precursor frequency of both Thl and Th2 cells. In lung tissue, we did not observe an increase in numbers of CD4+ T lymphocytes in mice inoculated with P. carinii but found a delayed and significant increase in precursor frequency for both Thl and Th2 cells.

The temporal lag in accumulation of Th1 and Th2 cells in lung tissue, compared with lymph nodes, is consistent with early proliferation of CD4+ T lymphocytes in lymph nodes as part of a primary immune response followed by subsequent recruitment of these cells to lung tissue. This concept is supported by previous studies with mice that showed that, after exposure to intratracheal antigen, specific immune cells form first in the regional lymph nodes and then are recruited into lung tissue [30]. However, our observations about P. carinii cannot exclude in situ proliferation as an additional mechanism for increased Th1/Th2 cells in lung tissue after intratracheal challenge with P. carinii.

In addition to a change in numbers of Th1 and Th2 cells in lung tissue, our data demonstrate that CD4+ T lymphocytes in infected lung tissue also may have an enhanced capacity to release specific cytokines. Early after intratracheal inoculation, CD4+ T lymphocytes in hilar lymph nodes have an enhanced capacity to release the Th2 cytokine IL-4, whereas the release of the Thl cytokine IFN-γ is enhanced later in lung tissue.

These observations are of interest because they may reflect the local cytokine milieu in the alveolar space during P. carinii infection. It is exposure to cytokines that probably provides the dominant differentiation stimulus for T lymphocytes' response to antigen. Cytokine products of one T cell subset may inhibit cell proliferation of the opposing subset, which polarizes the T cell population to a Thl or Th2 profile [31]. For example, IFN-γ inhibits the development of Th2 cells [32, 33], whereas IL-12, a product of macrophages and NK cells, both promotes proliferation of Th1 cells and suppresses proliferation of Th2 cells [14, 34]. Conversely, cytokine products of Th2 cells, such as IL-4, support proliferation of Th2 cells [35, 36], whereas other products, such as IL-10, suppress the release of IL-2 and IFN-γ by Thl cells [37]. Thus, the cytokine milieu within which CD4+ T lymphocytes are exposed to P. carinii may have an important modulatory influence on whether these cells differentiate into a Th1 or a Th2 lymphocyte subset.

In support of this idea, studies from our laboratory indicate that augmentation of IFN-γ in lung tissue through in vivo transfer of the IFN-γ gene increases the number of lung CD8+ T lymphocytes that release Th1 cytokines after inoculation with P. carinii [38]. However, these data do not prove the existence of an essential role for IFN-γ in the normal host response to P. carinii. Chen et al. [39] showed that antibody neutralization of IFN-γ did not prevent clearance of experimental infection.

Our data demonstrate that at least some of the T lymphocytes recruited to lung tissue can generate a specific in vitro proliferative response to P. carinii. T lymphocytes that respond specifically to P. carinii have been isolated and studied in vitro by other investigators [40, 41]. We have not been successful in adapting our ELISPOT assay to detect antigen-specific T lymphocytes recruited to the lungs of mice inoculated with P. carinii in vivo, perhaps because of the small numbers of such cells and the requirement for antigen-presenting cells in the cultures. Other investigators have shown that recruitment of antigenspecific lymphocytes into lung tissue during a variety of disease processes is accompanied by a more generalized recruitment of non-antigen-specific lymphocytes [42]. We feel that a more sensitive method than the ELISPOT assay technique will be necessary to calculate precursor frequency of P. carinii-specific CD4+ T lymphocytes in lung tissue.

Host defense against P. carinii is critically dependent on T lymphocytes, specifically CD4+ T lymphocytes. This has been demonstrated with a variety of approaches, including antibody depletion of CD4+ T lymphocytes [5, 6] and adoptive transfer of CD4+ cells into immunodeficient mice [7] and gene disruption mutant mice [8, 43]. CD8+ T lymphocytes are not sufficient by themselves to resolve experimental infection [6, 7, 43], although they may play a partial host-defense role in the setting of CD4+ T lymphocyte deficiency [44].

In contrast, little is known about the role of specific subsets of CD4+ T lymphocytes in host defense against P. carinii. Garvy et al. [45] addressed T lymphocyte subsets indirectly through analysis of IgG antibody subclasses in mice challenged with P. carinii. Their study design was different from ours in that mice first were inoculated with P. carinii and then were depleted of CD4+ T lymphocytes and rechallenged. Garvy et al. found that wild-type mice generated an IgG1 antibody response, which is consistent with a Th2 response. These antibodies were detectable at roughly the same time after challenge (3 weeks) that we observed maximal lymphocyte recruitment into lung tissue. Garvy et al. also observed that IL-4 knock-out mice could mount an antibody response of the Thl type and still resolve infection.

Our study is the first to isolate and characterize T lymphocyte subsets during the host response to this infectious pathogen. Our results are consistent with those of Garvy et al. [45] and indicate that both Th1 and Th2 subsets can participate in clearance of infection. Thus, lymphocyte responses to P. carinii, at least in the mouse, are not clearly dichotomous between Thl and Th2, as has been demonstrated for Leishmania species [16, 17]. Further definition of the host defense roles of specific T lymphocyte subsets for P. carinii will require adoptive transfer of polarized lymphocytes, research of which is underway.

Understanding how T lymphocytes participate in host defense against P. carinii is important not just with regard to the basic biology of P. carinii but with regard to HIV infection as well. HIV infection is characterized not just by a progressive decline in the number of circulating CD4+ T lymphocytes but also by alterations in T lymphocyte functions. Profound alterations in T lymphocyte function may be seen in HIV-infected persons with only modestly decreased numbers of CD4+ T lymphocytes. Epidemiologic studies of HIV-infected persons have shown an early decline in skin test reactivity (delayed hypersensitivity, Thl surrogate) followed by a later augmentation of antibody responses to vaccination (humoral immunity, Th2 surrogate) [46]. These observations have led to the theory that during the progression of HIV infection, there is a switch in lymphocyte responses from an early Th1- to a Th2-dominant pattern, followed by a loss of all T lymphocyte responses [47–49]. If our results, which show that both Th1 and Th2 lymphocyte responses are involved in clearance of P. carinii, can be extrapolated to humans, this may explain why P. carinii pneumonia is observed clinically only at an advanced stage of HIV infection when both Th1 and Th2 responses have been compromised.

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Footnotes

  • All animal procedures followed guidelines established by the Louisiana State University Health Sciences Center (LSUHSC) Institutional Animal Care and Use Committee.

  • Grant support: National Institutes of Health (HL-59724, AA-08845); LSUHSC Alcohol Research Center.

  • Received January 7, 2000.
  • Revision received March 6, 2000.
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  1. J Infect Dis. (2000) 181 (6): 2011-2017. doi: 10.1086/315487
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