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CD8⁺ T Cells Accumulate in the Lungs of Mycobacterium tuberculosis-Infected K^b-/-D^b-/- Mice, But Provide Minimal Protection ¹

Kevin B. Urdahl^*, Denny Liggitt and Michael J. Bevan^2,

Departments of ^* Pediatrics, Comparative Medicine, and Immunology, Howard Hughes Medical Institute, University of Washington, Seattle, WA 98195

	Abstract

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Recent studies have shown that MHC class I molecules play an important role in the protective immune response to Mycobacterium tuberculosis infection. Here we showed that mice deficient in MHC class Ia, but possessing MHC class Ib (K^b-/-D^b-/- mice), were more susceptible to aerosol infection with M. tuberculosis than control mice, but less susceptible than mice that lack both MHC class Ia and Ib (₂m^-/- mice). The susceptibility of K^b-/-D^b-/- mice cannot be explained by the failure of CD8⁺ T cells (presumably MHC class Ib-restricted) to respond to the infection. Although CD8⁺ T cells were a relatively small population in uninfected K^b-/-D^b-/- mice, most already expressed an activated phenotype. During infection, a large percentage of these cells further changed their cell surface phenotype, accumulated in the lungs at the site of infection, and were capable of rapidly producing IFN- following TCR stimulation. Histopathologic analysis showed widespread inflammation in the lungs of K^b-/-D^b-/- mice, with a paucity of lymphocytic aggregates within poorly organized areas of granulomatous inflammation. A similar pattern of granuloma formation has previously been observed in other types of MHC class I-deficient mice, but not CD8^-/- mice. Thus, neither the presence of MHC class Ib molecules themselves, nor the activity of a population of nonclassical CD8⁺ effector cells, fully restored the deficit caused by the absence of MHC class Ia molecules, suggesting a unique role for MHC class Ia molecules in protective immunity against M. tuberculosis.

	Introduction

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Natural infection by Mycobacterium tuberculosis (Mtb) ³ occurs by inhalation of aerosolized droplets containing bacilli. Bacteria are taken up by alveolar macrophages in the lung and replicate within endosomal vesicles. Within this intracellular environment, Mtb has evolved mechanisms to evade the immune system and can often survive the lifetime of the host. Although the immune system usually does not eliminate the infection, it is often successful in containing the bacteria, limiting tissue damage, and preventing respiratory compromise. This containment depends on the ability of T cells to infiltrate the lung and direct the appropriate differentiation and organization of immune effector cells into a structure known as a granuloma (1, 2).

The majority of T cells within Mtb-induced granulomas in the lung are CD4⁺ T cells (3). These CD4⁺ T cells recognize Mtb-derived Ags presented by MHC class II-positive cells, release cytokines such as IFN- and TNF-, and activate macrophages to exhibit antimycobacterial properties (2). CD4⁺ T cells are critical for immune protection, and mice deficient in MHC class II molecules (4, 5, 6) or CD4⁺ T cells (5) die rapidly following Mtb infection.

CD8⁺ T cells also infiltrate the lung following infection with Mtb, but only a few become part of the structure of the granuloma (3). Their relative importance in immune protection has been subject to controversy. Mice deficient in MHC class I as a result of disruption of the ₂-microglobulin (₂m) gene lack CD8⁺ T cells and are quite susceptible to infection (7) (although not as susceptible as class II^-/- mice (6)), a finding that was initially interpreted to reflect a critical role for CD8⁺ T cells. More recent studies have found that other mice lacking CD8⁺ T cells, but possessing MHC class I molecules (i.e., CD8 gene knockout mice (8, 9) or Ab-depleted mice (6)), are much less susceptible. This suggests that MHC class I molecules may contribute to immunity by mechanisms independent of CD8⁺ T cells. An intriguing report by D’Souza et al. (9), showed that ₂m^-/-, but not CD8^-/-, mice, fail to form lymphocyte aggregates normally associated with granulomatous inflammation in the lung.

Nevertheless, some experiments clearly show that CD8⁺ T cells can contribute to immunity against tuberculosis. Adoptive transfer of immune CD8⁺ T cells (10, 11) or immunization with Mtb epitopes recognized by CD8⁺ T cells can confer partial protection against subsequent challenge with Mtb (12, 13, 14, 15). Furthermore, a recent study using a mouse model of latency and reactivation suggests that CD8⁺ T cells may be even more important than CD4⁺ T cells in controlling the latent phase of tuberculosis infection (16). Therefore, despite the possibility that much of the susceptibility to active tuberculosis of MHC class I-deficient mice may be explained by CD8-independent immune mechanisms, the involvement of CD8⁺ T cells deserves further study, since they may play a critical role in controlling latent disease, which comprises the majority of human infection. Thus, targeting CD8⁺ T cells that recognize Mtb-derived Ags may still be an important consideration in vaccine design.

Both MHC Ia-restricted (or classical) and MHC class Ib-restricted (or nonclassical), Mtb-reactive CD8⁺ T cells have been identified in humans and mice (2), but their relative contributions of each to immunity are unclear. Unlike MHC class Ia-restricted T cells, MHC class Ib-restricted CD8⁺ T cells are constitutively activated in naive animals (17, 18) and respond rapidly to infectious challenge (17, 19). These unique properties of MHC class Ib-restricted CD8⁺ T cells may be inherent to this subclass of cells, because the phenotype begins to be expressed during development in the thymus and correlates with a distinct pathway of positive selection (18). The possible role of MHC class Ib-restricted CD8⁺ T cells during chronic bacterial infection, such as tuberculosis, is not known. In recently infected humans, Lewinsohn et al. (20, 21) showed that the vast majority of Mtb-reactive, CD8⁺ T cells in the peripheral blood are restricted by the MHC class Ib molecule, HLA-E. CD1-restricted CD8⁺ T cells have also been isolated from peripheral blood of infected individuals (22, 23, 24). In mice, Mtb-reactive CD8⁺ T cells restricted by the MHC class Ib molecule, H2-M3, have been identified (25). In addition, Rolph et al., reported that animals lacking MHC class Ia, but possessing MHC class Ib (K^b-/-D^b-/- mice), have an intermediate susceptibility to high dose, i.v. administered Mtb compared with wild-type and MHC class Ia- and Ib-deficient (₂m^-/-) mice (26). The CD8⁺ T cell response, however, was not investigated in this study, and the pulmonary immune response of MHC class Ib-restricted CD8⁺ T cells during tuberculosis infection has not yet been examined.

To address this issue, we studied the changes in CD8⁺ T cells in lungs and draining lymph nodes of K^b-/-D^b-/- mice over a 6-mo period following low dose, aerosol infection with Mtb. Because K^b-/-D^b-/- mice lack MHC class Ia molecules, but possess MHC class Ib molecules and a small population of CD8⁺ T cells (a population that is absent in ₂m^-/- mice that lack both MHC class Ia and Ib molecules), it is reasonable to assume that most CD8⁺ T cells in these mice are MHC class Ib restricted. Our results show that CD8⁺ T cells in K^b-/-D^b-/- mice are highly recruited to the lungs following infection. They undergo phenotypic and functional changes indicative of maturation into effector cells. Interestingly, these nonclassically restricted CD8⁺ T cells display a different phenotype and contain a higher percentage of activated cells, both before and after infection, than classically restricted CD8⁺ T cells in wild-type mice. We also found, similar to a previous report that used i.v. infection, that K^b-/-D^b-/- mice infected by aerosolization were significantly more susceptible to disease than wild-type mice and were only slightly more resistant than ₂m^-/- mice (26). In addition, similar to the findings of D’Souza et al. (9) in ₂m^-/- mice, K^b-/-D^b-/- mice had defective pulmonary granulomas with diminished lymphocyte aggregation. Therefore, despite the presence of a population of highly activated CD8⁺ T cells in lungs of K^b-/-D^b-/- mice, the absence of MHC class Ia molecules still leads to an inability to control infection with Mtb.

	Materials and Methods

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Mice

C57BL/6, ₂m^-/- (backcrossed 11 times to the C57BL/6 background), TAP-1^-/- (backcrossed 10 times to C57BL/6), and CD8^-/- mice (backcrossed 13 times to C57BL/6) were purchased from The Jackson Laboratory (Bar Harbor, ME). K^b-/-D^b-/- breeders, backcrossed six times to C57BL/6, were obtained from J. Forman (University of Texas Southwestern Medical Center, Dallas, TX). K^b-/-D^b-/- mice were bred and maintained in our facility at the University of Washington. Mice were used between 6 and 8 wk of age. All components of the experiments with animals were approved by the animal care committee at the University of Washington and comply with federal guidelines.

Bacteria and aerosol infections

Mtb H37Rv strain (ATCC 25618; American Type Culture Collection, Manassas, VA) was initially passed once through mice. A single CFU was selected from lung tissue, inoculated in Proskauer-Beckett liquid medium (27), and grown to mid-log phase, and individual aliquots were frozen at -80°C. An aliquot of frozen mycobacteria was thawed and briefly sonicated before each infection to disrupt any clumps of bacteria. Mice were infected with a predetermined infective dose using an airborne infection apparatus, such that 100 viable bacteria were delivered to the lungs of each mouse (28). The number of viable bacteria in the lungs was determined at various time points by plating serial dilutions of lung homogenates on Middlebrook 7H10 agar and counting colonies after 3 wk at 37°C. A day 1 count was performed to determine the infecting dose. At the same time as in morbidity experiments, mice were weighed weekly and euthanized when their weight dropped by 20%. This degree of weight loss was associated with signs of morbidity, including respiratory distress, hunched posture, and lack of grooming.

NK cell depletion

C57BL/6 mice were given injections of 200 µg of anti-NK1.1 Ab (PK-136; American Type Culture Collection) on days -2, 0, 3, 6, 9, and 12 postinfection with Mtb via the aerosol route. The efficacy of depletion was determined on days 7 and 14 by staining spleen and lung cells with anti-DX5-PE (BD PharMingen, San Diego, CA) and anti-CD3-FITC (clone 145-2C11; BD PharMingen) and analyzing by flow cytometry. NK cells were identified as the DX5⁺CD3^- cells within these populations.

Antibodies

The following mAbs were used for flow cytometry: anti-CD11a FITC (clone 2D7; BD PharMingen), anti-CD44 FITC (clone IM7; BD PharMingen), anti-CD49d (₄ integrin) FITC (clone R1-2; BD PharMingen), anti-CD62L FITC (clone MEL-14; BD PharMingen), anti-CD69 FITC (clone H1.2F3; BD PharMingen), anti-CD122 FITC (clone TM-1; BD PharMingen), anti-Ly6C FITC (clone AL-21; BD PharMingen), anti-TCR -chain (clone H37-597; BD PharMingen), anti-₇ integrin PE (clone M293; BD PharMingen), biotin-conjugated anti-CD29 (₁ integrin; clone Ha2/5; BD PharMingen) anti-CD4-CyChrome (clone RM4-5; BD PharMingen), and anti-CD8-allophycocyanin (clone 53-6.7; BD PharMingen). Streptavidin conjugated with FITC (Vector Laboratories, Burlingame, CA) or PE (Molecular Probes, Eugene, OR) was used as a secondary reagent for biotin-labeled Abs. Flow cytometry was conducted on a FACSCalibur and analyzed using CellQuest software (BD Biosciences, Mountain View, CA).

Preparation of single-cell suspensions from lung and lymph nodes

Intraparenchymal lung lymphocytes were isolated by a modified procedure developed by Abraham et al. (29). Mice were sacrificed by cervical dislocation. Lungs were perfused with PBS containing heparin, minced, and incubated for 30 min with shaking in 1 ml/lung of 1 µg/ml collagenase V/dispase (Sigma-Aldrich, St. Louis, MO) and 50 µg/ml DNase I (Invitrogen, Carlsbad, CA) in PBS. Intact lung tissue was disrupted by passage through an 18-gauge needle and filtered through a 100-µm pore size cell strainer (BD Biosciences, Franklin Lakes, NJ). Cells were washed once with complete medium, resuspended in 4 ml of 40% Percoll (Pharmacia Biotech, Uppsala, Sweden), and layered over 80% Percoll. After centrifugation at 600 x g for 20 min at 15°C, the cells at the interface were collected and washed twice in complete medium. Remaining red cells were lysed in a hypotonic buffer. A single-cell suspension of lymph node cells was obtained by passing teased lymph nodes through a 100-µm strainer and lysing red cells using a hypotonic buffer.

Intracellular IFN- staining

Wells of 24-well plates were precoated with 50 µg/ml of anti-hamster IgG (clone G94-90.5; BD PharMingen) for 1 h at 37°C, washed three times with PBS, and coated overnight with 5 µg/ml of anti-CD3 (clone 145-2C11; BD PharMingen) at 4°C. Intracellular IFN- staining was performed using a kit as instructed by the manufacturer (BD PharMingen). Briefly, lung or lymph node cells, prepared as previously described, were stimulated with plate-bound anti-CD3 for 5 h in complete RPMI (RPMI 1640 supplemented with 10% FCS, 2 mM L-glutamine, 10 mM HEPES, 0.5 µM 2-ME, 100 U/ml penicillin, and 100 µg/ml streptomycin) in the presence of brefeldin A. Cells were washed, stained with anti-CD8-allophycocyanin, resuspended in permeabilization-fixation buffer, and stained with anti-IFN- FITC (clone XMG1.2; BD PharMingen). Labeled cells were washed in permeabilization buffer, resuspended in fix buffer, and analyzed on a FACSCalibur.

Histology

The lower lobe of the right lung of each mouse was inflated with methyl Carnoys (60% methanol, 30% chloroform, and 10% glacial acetic acid) and fixed overnight. Organs were embedded in paraffin, sectioned, and stained with H&E or a modified Fite stain. Slides were examined without knowledge of experimental group and were subjectively graded for quantity and quality of cellular accumulation.

	Results

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Role of MHC class Ia and class Ib molecules in immunity to Mtb

To test the role of MHC class Ia molecules in immunity against Mtb, we infected K^b-/-D^b-/- mice with a low dose of Mtb via the aerosol route. Because mice of the H2^b background do not express H2-L, K^b-/-D^b-/- mice have a complete absence of MHC class Ia molecules, but express MHC class Ib molecules normally. For comparison, C57BL/6 and ₂m^-/- mice (deficient in both MHC class Ia and Ib molecules) were infected as controls. The bacterial loads in the lungs from 7–177 days after infection are shown in Fig. 1. Interestingly, both ₂m^-/- and K^b-/-D^b-/- mice showed higher bacterial burdens at early time points after infection; they had 0.5 logs more bacteria in their lungs than C57BL/6 mice on days 7 and 14 postinfection. Because these differences were small, we repeated these early time points and found that MHC class I-deficient mice (both ₂m^-/- and K^b-/-D^b-/- mice) had statistically more bacteria in their lungs in five of five experiments analyzed between 6 and 14 days after infection (Figs. 1 and 2, and data not shown). To determine whether these apparent differences in innate immunity were due to the absence of MHC class Ia-restricted CD8⁺ T cells or to the absence of MHC class I molecules, we examined another type of MHC class I-deficient mice (TAP-1^-/- mice) and mice deficient in CD8 T cells, but with normal MHC class I expression (CD8^-/- mice). We found that TAP-1^-/-, but not CD8^-/-, mice, also had increased bacterial load in the lungs at early time points after infection (Fig. 2). These data suggest that MHC class Ia molecules may regulate early phases of immunity against Mtb by a mechanism that is independent of CD8⁺ T cells.

View larger version (27K): [in this window] [in a new window]   FIGURE 1. Bacterial burden in the lung after aerosol infection with Mtb. C57BL/6 (), 2m-/- (), and Kb-/-Db-/- () mice were infected with 100 CFU of Mtb via the aerosol route. Bacterial load in the lung was measured on days 7, 14, 21, 42, 85, and 177 postinfection. Each symbol represents the bacterial burden of an individual mouse in each group, and the mean for each group is shown. Because our infection chamber allows only 40 mice to be infected in a single experiment, data from three independent infections are shown (e.g., days 7 and 85 were generated from one infection, days 14 and 177 from a second infection, and days 21 and 42 from a third infection). In each of these three infections, the infective dose was verified as 100 CFU on day 1 postinfection. Statistical differences between C57BL/6 and 2m-/- mice were present on day 7 (p < 0.05), day 14 (p < 0.05), day 42 (p < 0.01), day 85 (p < 0.001), and day 177 (p < 0.001). Statistical differences between C57BL/6 and Kb-/-Db-/- mice were present on day 7 (p < 0.05), day 14 (p < 0.05), day 85 (p < 0.001), and day 177 (p < 0.01). Statistical differences between 2m-/- and Kb-/-Db-/- mice were present on day 42 (p < 0.01) and day 85 (p < 0.01).

View larger version (18K): [in this window] [in a new window]   FIGURE 2. Impaired innate immunity in MHC class I-deficient mice after aerosol infection with Mtb. C57BL/6 (), 2m-/- (), TAP-1-/- (), and CD8-/- () mice were infected with 50 CFU of Mtb via the aerosol route. Bacterial load in the lung was measured on day 8 postinfection. Each symbol represents the bacterial burden of an individual mouse in each group, and the mean for each group is shown. Statistical differences between C57BL/6 and 2m-/- (p < 0.05) and TAP-1-/- (p < 0.05) mice were present.

Despite these early differences in innate immunity, at 21 days postinfection there were no differences between any of the groups, with each reaching 6 log bacteria in the lung (Fig. 1). The bacterial load in C57BL/6 mice remained at this level for >6 mo, indicating effective control of infection by the adaptive immune system in C57BL/6 mice. In contrast, the bacterial load in ₂m^-/- mice continued to rise and reached a plateau by day 85 that was over 1.5 logs higher than the plateau in C57BL/6 mice. By comparison, the rise in the bacterial load in the lungs of K^b-/-D^b-/- mice was delayed. At 42 days, K^b-/-D^b-/- mice were identical with C57BL/6 mice, but started to exhibit a higher load by day 85, and by day 177 had as many bacteria in their lungs as ₂m^-/- mice.

We also monitored the time to morbidity of C57BL/6 mice, ₂m^-/- mice, and K^b-/-D^b-/- mice following low dose aerosol infection as a further measurement of their protective immunity. The results were consistent with the pulmonary bacterial loads in these mice. ₂m^-/- mice were most susceptible to infection, with a mean time to morbidity of 176 ± 30 days, whereas C57BL/6 mice showed a time to morbidity of 385 ± 46 days (Fig. 3). K^b-/-D^b-/- mice exhibited slightly more resistance to infection than ₂m^-/- mice with a time to morbidity of 242 ± 56 days. Similarly, Rolph et al. (26) recently showed that K^b-/-D^b-/- mice had an intermediate phenotype of susceptibility between C57BL/6 and ₂m^-/- mice following high dose i.v. infection. Thus, the presence of MHC class Ib fails to fully compensate for the absence of MHC class Ia in K^b-/-D^b-/- mice.

View larger version (27K): [in this window] [in a new window]   FIGURE 3. Decreased time to morbidity of MHC class I-deficient mice after aerosol infection with Mtb. C57BL/6 (), 2m-/- (), and Kb-/-Db-/- () mice were infected with 100 CFU of Mtb via the aerosol route. Data pooled from two independent experiments, with 12–13 mice/group, are shown. Differences between each group were statistically significant; C57BL/6 vs 2m-/- mice (p < 0.0001), C57BL/6 vs Kb-/-Db-/- mice (p < 0.0001), and 2m-/- (p < 0.0001) vs Kb-/-Db-/- mice.

If MHC class Ib-restricted CD8⁺ T cells did not participate in the pulmonary immune response against Mtb, this might help explain why K^b-/-D^b-/- mice were still susceptible to tuberculosis. To investigate this possibility, we examined the T cells in paratracheal lymph nodes and those infiltrating the lung at various time points following aerosol infection. In uninfected K^b-/-D^b-/- mice, CD8⁺ T cells comprised a minor population of the total lymph node cells (1–3%; data not shown). After aerosol infection, the number of total cells in the paratracheal lymph node increased up to 50-fold (data not shown); however, the percentage of CD8⁺ T cells within the lymph nodes remained essentially the same (1–3%) at all time points examined (days 7–189 postinfection; data not shown).

CD8⁺ T cells also were a relatively small subset of the cells infiltrating the lungs of uninfected K^b-/-D^b-/- mice (Fig. 5A); only 10% as many CD8⁺ T cells were present in the lungs of K^b-/-D^b-/- mice compared with C57BL/6 mice (Figs. 4 and 5A). After infection, however, an expanded population of CD8⁺ T cells was observed in the lungs of K^b-/-D^b-/- mice. As a consequence of this expansion in the lungs, the CD4/CD8 ratio of the infiltrating T cells changed from 20:1 in uninfected K^b-/-D^b-/- mice to 3:1 in Mtb-infected mice (Fig. 5, A and B). In fact, at late time points after Mtb infection, K^b-/-D^b-/- mice accumulated almost as many (or more) total CD8⁺ T cells in their lungs (ranging from 50–120%) as C57BL/6 mice (Fig. 4). These results suggest that MHC class Ib-restricted CD8⁺ T cells are heavily recruited to the lungs following aerosol infection with Mtb.

View larger version (33K): [in this window] [in a new window]   FIGURE 5. CD8+ T cells accumulate in the lungs of Kb-/-Db-/- mice after aerosol infection with Mtb. A, Lung cells from uninfected C57BL/6, 2m-/-, and Kb-/-Db-/- mice were analyzed by flow cytometry for the expression of TCR vs CD8 or CD4. B, Lung cells from Mtb-infected (74 days postinfection) C57BL/6, 2m-/-, and Kb-/-Db-/- mice were analyzed by flow cytometry for expression of TCR vs CD8 or CD4. The percentages of TCR+CD4+ cells and TCR+CD8+ cells within total lung cell populations are shown. Similar results were obtained in eight independent experiments.

View larger version (19K): [in this window] [in a new window]   FIGURE 4. T cells accumulate in the lungs after aerosol infection with Mtb. C57BL/6, 2m-/-, and Kb-/-Db-/- mice were infected with 100 CFU of Mtb via the aerosol route. The numbers of CD4+ () and CD8+ () T cells in the lungs (x10-5) were calculated on days 0, 21, 77, and 189 postinfection by multiplying the number of lung cells isolated from collagenase-treated lungs by the percentage of TCR+CD4+ or TCR+CD8+ cells found in this population, as determined by flow cytometry.

CD4⁺ T cells also infiltrated the lungs of both C57BL/6 and MHC class I-deficient mice after aerosol infection (Figs. 4 and 5B). After an initial influx of T cells into the lungs 3 wk after infection, the number of intrapulmonary CD4⁺ T cells in C57BL/6 mice remained relatively constant, increasing only slightly at 6 mo (Fig. 4). In contrast, the number of intrapulmonary CD4⁺ T cells in ₂m^-/- mice continued to increase throughout the course of the infection. K^b-/-D^b-/- mice showed an intermediate phenotype; the CD4⁺ T cells increased in number in the lungs, but to a lesser extent than seen in ₂m^-/- mice. Thus, although infiltration of the lung with Mtb-reactive T cells is necessary for effective containment of the infection, after effective containment is achieved (as occurs in C57BL/6 mice), an equilibrium is reached in which the numbers of both bacteria (Fig. 1) and T cells (Fig. 4) in the lung remain relatively constant. However, if the immune response is unable to control the infection (as occurs in MHC class I-deficient mice), T cell numbers continue to increase within the lungs.

We also examined the activation status of the T cells in the paratracheal lymph nodes and lungs during the course of Mtb infection (Table I). CD8⁺ T cells in K^b-/-D^b-/- mice exhibit a distinct activated phenotype even in uninfected mice. The majority of CD8⁺ T cells in the lymph nodes and lungs of uninfected K^b-/-D^b-/- mice were CD11a^high, CD44^high, CD122^high, Ly6C^high, ₁ integrin^high, and ₇ integrin^- (Table I and data not shown). Although some activation markers, such as CD44 and CD11a, were expressed at high levels on CD8⁺ T cells before infection and did not markedly change after infection, changes in the expression patterns of other activation markers were observed. For example, most CD8⁺ T cells in the lymph nodes and lungs of K^b-/-D^b-/- mice became CD122^- after infection. Despite the expression of this panel of activation markers in uninfected K^b-/-D^b-/- mice, most lymph node CD8⁺ T cells were CD62L⁺ (the naive phenotype), but activated CD62L^- CD8⁺ T cells became the predominant phenotype after infection. Furthermore, although most lung CD8⁺ T cells were CD69^- before infection, most became CD69⁺ after infection. Thus, although most CD8⁺ T cells in K^b-/-D^b-/- mice (presumably MHC class Ib restricted) had an activated phenotype before infection, the majority of CD8⁺ T cells in both lymph node and lung changed their phenotype even further after Mtb infection.

View this table: [in this window] [in a new window]   Table I. Percentage of TCR+CD8+ T cells in the paratracheal lymph node and lung at various time points after Mtb infection expressing a panel of cell surface activation phenotypes

Perhaps the most dramatic difference we observed between an activation marker on CD8⁺ T cells in C57BL/6 vs K^b-/-D^b-/- mice was in expression levels of ₄ integrin, a cell surface protein that pairs with ₁ integrin to form very late Ag-4 (30). Whereas, CD8⁺ T cells in both the lymph nodes and lungs of uninfected C57BL/6 mice expressed intermediate levels of ₄ integrin (Fig. 6), uninfected K^b-/-D^b-/- mice contained a large population of ₄ integrin^low CD8⁺ T cells in their lymph nodes and bimodally distributed populations of ₄ integrin^low and ₄ integrin^high CD8⁺ T cells in their lungs. After Mtb infection, CD8⁺ T cells in both the lymph nodes and lungs of C57BL/6 mice showed only slight increases in the percentages of ₄ integrin^high cells, but the phenotype of most CD8⁺ T cells in K^b-/-D^b-/- mice changed to ₄ integrin^high.

View larger version (29K): [in this window] [in a new window]   FIGURE 6. CD8+ T cells in C57BL/6 and Kb-/-Db-/- mice express different levels of 4 integrin. Surface expression of 4 integrin gated on TCR+CD8+ cells in the paratracheal lymph nodes or lungs, of uninfected or Mtb-infected (74 days postinfection) C57BL/6 or Kb-/-Db-/- mice. The percentages of 4 integrinlow and 4 integrinhigh cells within the TCR+CD8+ populations are shown. Similar results were obtained in three independent experiments.

A high percentage of MHC class Ib-restricted CD8⁺ T cells in the lung is capable of producing IFN-

We also compared the abilities of lung CD8⁺ T cells in C57BL/6 and K^b-/-D^b-/- mice to produce IFN- following TCR stimulation for 5 h in vitro (Fig. 7). CD8⁺ T cells isolated from the lungs of uninfected C57BL/6 mice were unable to produce IFN- after TCR stimulation, but after infection a few (8.9% of CD8⁺ T cells in the lung) were able to produce IFN-. In contrast, a small number of CD8⁺ T cells (9.2%) in the lungs of K^b-/-D^b-/- mice were able to produce IFN- even before infection. After Mtb infection, this number increased markedly to 25.1% in K^b-/-D^b-/- mice. Thus, consistent with our previous observations on the preactivated phenotype of CD8⁺ T cells in K^b-/-D^b-/- mice (18), K^b-/-D^b-/- mice contain more IFN--producing, effector CD8⁺ T cells than C57BL/6 mice before infection and an even more marked increase in these cells after infection. In contrast, similar percentages of CD4⁺ T cells in Mtb-infected C57BL/6, ₂m^-/-, and K^b-/-D^b-/- mice were capable of producing IFN- (data not shown).

View larger version (31K): [in this window] [in a new window]   FIGURE 7. Mtb infection induces a large population of IFN--producing CD8+ T cells in Kb-/-Db-/- mice. Lung cells from uninfected or Mtb-infected (day 59 postinfection) mice were stimulated with medium or plate-bound anti-CD3, and intracellular IFN- was analyzed by flow cytometry. The percentages of IFN--expressing cells within the CD8+ population are shown.

Next, we compared the histopathologic changes in lung tissues from C57BL/6, ₂m^-/-, and K^b-/-D^b-/- mice following aerosol infection with Mtb. By 42 days postinfection differences between C57BL/6 mice and MHC class I-deficient mice became clearly evident (Fig. 8). C57BL/6 mice had mostly normal lung parenchyma, with a few scattered foci of well-organized granulomatous lesions. The central core of these lesions contained macrophages, many of which were infected with Mtb when examined by modified Fite staining (data not shown). These infected macrophage were surrounded by infiltrating lymphocytes. The circumference of the lesions also contained macrophages; however, in contrast to the macrophages in the center of the lesions, most of these were uninfected (data not shown). In contrast, lungs from MHC class I-deficient mice (both ₂m^-/- and K^b-/-D^b-/- mice) contained more widespread inflammatory lesions (involving at least 2–3 times more of the lung parenchyma compared with C57BL/6 mice). Although a few areas of lymphocyte aggregation were observed in the macrophage fields in both ₂m^-/- and K^b-/-D^b-/- mice at 42 days postinfection, the lesions were less organized and contained more degenerative, foamy macrophages, necrotic cells, and neutrophils than the lesions observed in C57BL/6 mice. Finally, acid-fast organisms were not contained within the center of the granulomatous lesions in MHC class I-deficient mice, but were more widely dispersed throughout the inflammatory macrophage fields. Importantly, despite these marked differences in histopathology, C57BL/6 and K^b-/-D^b-/- mice (at 42 days postinfection) had the same number of bacteria in their lungs (Fig. 1). Thus, the inability to form a well-organized granuloma precedes the increased bacterial burden in the lungs of K^b-/-D^b-/- mice.

View larger version (87K): [in this window] [in a new window]   FIGURE 8. MHC class I-deficient mice have aberrant granuloma formation in the lungs after aerosol infection with Mtb. Representative lung sections from C57BL/6, 2m-/-, and Kb-/-Db-/- mice at 42 days (magnification, x25) and 85 days (magnification, x10) postinfection. The dark-staining cells in these photographs are lymphocytes with the paler-staining cells being primarily macrophages. Notice in the C57BL/6 mice the coalescing of lesions and the enhanced lymphocyte aggregation, particularly by day 85. In contrast, in both the 2m-/- and Kb-/-Db-/- mice, lesions tend to evolve little structurally, are composed of diffuse sheets of macrophages, and have a paucity of distinct lymphoid aggregates.

	Discussion

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Although MHC class Ib-restricted CD8⁺ T cells specific for Mtb-derived Ags have been isolated from infected mice and humans, their role in immunity against Mtb is unclear. This study represents the first attempt to characterize the pulmonary immune response of MHC class Ib-restricted CD8⁺ T cells following aerosol infection with Mtb. We showed that a large population of highly activated CD8⁺ T cells accumulated in the lungs of K^b-/-D^b-/- mice after infection. The presence of these CD8⁺ T cells in the lungs of K^b-/-D^b-/- mice, however, did not markedly improve resistance against aerosol infection with Mtb; although K^b-/-D^b-/- mice were slightly more resistant to infection than ₂m^-/- mice, they were more susceptible to infection that C57BL/6 mice.

Previous studies have shown that MHC class Ib-restricted CD8⁺ T cells exhibit a more activated phenotype than MHC class Ia-restricted CD8⁺ T cells in uninfected mice (17, 18). Here we showed, in addition, that CD8⁺ T cells in K^b-/-D^b-/- mice differ in their response to Mtb infection. Whereas only a minority of CD8⁺ T cells in C57BL/6 mice (most of which are MHC class Ia restricted) converted to an activated phenotype after infection, the majority of CD8⁺ T cells in K^b-/-D^b-/- mice (which are presumably MHC class Ib restricted) changed their phenotype. This was true not only in the lung, but also in the draining lymph node, suggesting that a large percentage of MHC class Ib-restricted CD8⁺ T cells was involved in the response to Mtb. It is possible that some of these CD8⁺ T cells that change their phenotype may not truly be specific for Mtb-derived Ags, but could be specific for self-Ags that are expressed at higher levels under conditions of stress, such as in the lung after Mtb infection. This possibility would be consistent with the idea that some MHC class Ib-restricted T cells function to recognize and regulate host cells under conditions of stress (31, 32). On the other hand, MHC class Ib-restricted CD8⁺ T cells specific for Mtb-derived Ags have been identified (20, 21, 22, 23, 24, 25), and Lewinsohn et al. (21) have shown that the vast majority of Mtb-specific, CD8⁺ T cells in the peripheral blood of recently infected humans are MHC class Ib-restricted. Thus, it is possible that many of the CD8⁺ in K^b-/-D^b-/- mice that change their phenotype after Mtb infection may truly be specific for Mtb-derived Ags.

Previous studies have shown that MHC class Ib-restricted CD8⁺ T cells have many properties usually associated with innate effector cells. In addition to being constitutively activated (17, 18), they respond rapidly to acute infection, but participate minimally in the memory response to Listeria monocytogenes (17, 19). How these T cells respond over time to a chronic bacterial infection, such as tuberculosis, had not been previously examined. Interestingly, we found that CD8⁺ T cells in K^b-/-D^b-/- mice continued to increase in number in the lungs during chronic infection. The latest time point we examined (day 189 postinfection) had the highest absolute number of CD8⁺ T cells in the lungs (Fig. 3). Furthermore, this late time point had the highest percentage of CD8⁺ T cells capable of producing IFN- after anti-CD3 stimulation (data not shown; 26.8 and 39.8% of CD8⁺ T cells in the paratracheal lymph nodes and lungs, respectively, of K^b-/-D^b-/- mice vs 8.1 and 26.6% in C57BL/6 mice). Therefore, in addition to being innate-like T cells, MHC class Ib-restricted CD8⁺ T cells have the ability to persist and continue to increase their effector function during chronic infection with Mtb.

Despite the accumulation of a large number of activated, IFN--producing, CD8⁺ T cells in the lung, K^b-/-D^b-/- mice are only slightly more resistant to infection than ₂m^-/- mice that have very few CD8⁺ T cells. Some investigators have suggested that aberrant iron metabolism in ₂m^-/- mice (due to lack of expression of the hereditary hemochromatosis gene, an MHC class I-like molecule involved in iron transport (33)) contributes to their increased susceptibility to infection with Mtb (34). If so, this would serve to diminish the contribution of MHC class Ib-restricted CD8⁺ T cells even further, because K^b-/-D^b-/- mice have normal iron metabolism. One possible explanation for the failure of MHC class Ib-restricted CD8⁺ T cells to fully restore protective immunity against Mtb might be that MHC class Ia-restricted CD8⁺ T cells are uniquely equipped with effector functions that are unable to be performed by MHC class Ib-restricted CD8⁺ T cells. Such differences are not currently appreciated; both types of CD8⁺ T cells (including those specific for Mtb-derived Ags) produce IFN- and can mediate cellular cytotoxicity. Furthermore, unlike the scenario presented here for Mtb, MHC class Ib-restricted CD8⁺ T cells have the ability to compensate for the absence of MHC class Ia-restricted CD8⁺ T cells and provide protective immunity against L. monocytogenes (17, 35).

A different explanation for the failure of large numbers of CD8⁺ T cells in K^b-/-D^b-/- mice to restore resistance against Mtb is suggested by the observation that CD8-deficient mice are less susceptible to Mtb infection than MHC class I-deficient mice (6, 8, 9). These studies suggest that MHC class I molecules may regulate immunity against Mtb by a mechanism that is independent of CD8⁺ T cells. We have confirmed the observations of D’Souza et al. (9) that, unlike MHC class I-deficient mice (i.e., ₂m^-/- and TAP-1^-/- mice) that have poorly organized granulomas with a paucity of lymphocyte aggregates, CD8^-/- mice form normal-appearing granulomas that effectively contain the infection (Fig. 8 and data not shown). Here we extend these observations by showing that K^b-/-D^b-/- mice, like ₂m^-/- and TAP^-/- mice, form aberrant granulomas with very few lymphocyte aggregates, suggesting a role for MHC class Ia molecules in this process. In other words, the failure to form effective granulomas correlates better with the lack of MHC class Ia molecules than with the lack of CD8⁺ T cells. These results suggest that MHC class Ia molecules themselves may be more important than CD8⁺ T cells in mediating granuloma formation and containment of Mtb infection.

How could MHC class Ia molecules regulate immunity against Mtb independently of CD8⁺ T cells? In addition to TCRs on CD8⁺ T cells, the immune system contains several other families of receptors that recognize MHC class I molecules. These receptors have been most well studied on NK cells, but are also expressed on macrophages, dendritic cells, B cells, and T cells (36). Most of these MHC class I-specific regulatory receptors deliver inhibitory signals to cells, and a smaller number deliver activating signals. On NK cells, these inhibitory receptors prevent NK cells from attacking uninfected host cells that express MHC class I molecules (36), but little is known about the role of these receptors in regulating the activity of other immune effector cell types.

We found that innate immunity against Mtb at very early time points was impaired in ₂m^-/-, TAP-1^-/-, and K^b-/-D^b-/- mice, but not in CD8^-/- mice. These findings suggest that innate immunity against Mtb is regulated by MHC class Ia molecules by a mechanism independent of CD8⁺ T cells. We suggest that implicating a role for MHC class Ia-specific regulatory receptors in regulating immunity against Mtb might be the most likely explanation for these observations. A role for NK T cells seems unlikely, because others have found no discernible difference between the immune response of CD1d-deficient and C57BL/6 mice against Mtb (37), suggesting that NK T cells do not play a significant role in immunity. In addition, our finding that K^b-/-D^b-/- mice have an impaired innate immune response despite having normal numbers of NK T cells argues against their role. Our experiments with NK-depleted C57BL/6 mice would seem to argue against a role for NK cells in this effect; however, this possibility cannot be completely excluded, because we were unable to achieve depletion of 100% of the NK cells in these experiments. It is also possible that MHC class I molecules regulate other cell types, such as macrophages, via MHC class I-specific regulatory receptors, and this possibility deserves further investigation.

We further hypothesize that MHC class Ia molecules, independent of CD8⁺ T cells, play a role in regulating adaptive immune processes required for granuloma formation and containment of Mtb infection in the lung. One possible receptor that may be involved in regulating adaptive immune responses is the inhibitory receptor PIR-B, a member of the Ig receptor superfamily (38). The closest relatives to PIR-B in humans, leukocyte Ig-like receptors (LIR) (also known as Ig-like transcripts), are encoded on human chromosome 19 in a position that is syntenic to the position of PIR-B in mice (38, 39). Like PIR-B, LIR are expressed primarily on APC, including macrophages, dendritic cells, and B cells (40, 41). The ligands for LIR are class I MHC molecules, and recognition of MHC class I by these inhibitory receptors on human dendritic cells has been implicated in regulating T cell activation and tolerance induction (42, 43). Although the ligand for PIR-B is still unclear, some recent evidence suggests that PIR-B may also recognize MHC class I molecules (44). Furthermore, PIR-B^-/- mice have recently been generated and found to have defective dendritic cell maturation and altered T cell responses (45). Although other MHC class I-specific receptors may also regulate immune responses against Mtb, PIR-B seems like a good candidate for further investigation.

Our data show that MHC class Ia-deficient mice have increased susceptibility to Mtb despite a large population of activated, CD8⁺ T cells in their lungs. Although CD8⁺ T cells may participate in some aspects of immunity against Mtb, perhaps, particularly in controlling latent infection, we believe that much of the susceptibility of MHC class I-deficient mice may be explained by CD8⁺ T cell-independent mechanisms. In the past ten years, since the first report of increased susceptibility to Mtb infection in ₂m^-/- mice (7), much has been learned about the presence of immunoregulatory receptors that recognize MHC class I molecules (36). We believe that understanding how these receptors regulate the complex immune processes that lead to granuloma formation and containment of Mtb in the lung will provide important insights into the role of MHC class I-specific receptors in regulating adaptive immunity and host defense.

	Acknowledgments

 
We thank Sherilyn Smith and Chris Wilson for establishing the pathogen level 3 facility and teaching us to perform aerosol infection with Mtb, Ethan Ojala for excellent technical assistance, Janie Yamagiwa for help with manuscript preparation, and Joke den Haan and Chris Wilson for critical reading of the manuscript.

	Footnotes

 
¹ This work was supported by National Institutes of Health Grants K12HD00850 from the Pediatric Scientist Development Program and T32A107411 (to K.B.U), the Howard Hughes Medical Institute, and National Institutes of Health Grant A119335 (to M.J.B.).

² Address correspondence and reprint requests to Dr. Michael J. Bevan, Howard Hughes Medical Institute, University of Washington, Box 357370, Seattle, WA 98195-7370. E-mail address: mbevan{at}u.washington.edu

³ Abbreviations used in this paper: Mtb, Mycobacterium tuberculosis; ₂m, ₂-microglobulin; LIR, leukocyte Ig-like receptor.

Received for publication August 19, 2002. Accepted for publication December 16, 2002.

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