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Infect Immun. Aug 2003; 71(8): 4297–4303.
PMCID: PMC166030

Investigation of the Role of CD8+ T Cells in Bovine Tuberculosis In Vivo

Abstract

Mycobacterium bovis is the causative agent of bovine tuberculosis (TB), and it has the potential to induce disease in humans. CD8+ T cells (CD8 cells) have been shown to respond to mycobacterial antigens in humans, cattle, and mice. In mice, CD8 cells have been shown to play a role in protection against mycobacterial infection. To determine the role of CD8 cells in bovine TB in vivo, two groups of calves were infected with the virulent M. bovis strain AF2122/97. After infection, one group was injected with a CD8 cell-depleting monoclonal antibody (MAb), and the other group was injected with an isotype control MAb. Immune responses to mycobacterial antigens were measured weekly in vitro. After 8 weeks, the animals were killed, and postmortem examinations were carried out. In vitro proliferation responses were similar in both calf groups, but in vitro gamma interferon (IFN-γ) production in 24-h whole-blood cultures was significantly higher in control cattle than in CD8 cell-depleted calves. Postmortem examination showed that calves in both groups had developed comparable TB lesions in the lower respiratory tract and associated lymph nodes. Head lymph node lesion scores, on the other hand, were higher in control calves than in CD8 cell-depleted calves. Furthermore, there was significant correlation between the level of IFN-γ and the head lymph node lesion score. These experiments indicate that CD8 cells play a role in the immune response to M. bovis in cattle by contributing to the IFN-γ response. However, CD8 cells may also play a deleterious role by contributing to the immunopathology of bovine TB.

Mycobacterium bovis is the causative agent of bovine tuberculosis (TB). It is also responsible for a portion of human TB cases, particularly in developing countries, where there are no control programs for bovine TB and the risk of opportunistic infection with M. bovis is increased by infection with human immunodeficiency virus (6). Thus, infection of cattle with M. bovis constitutes a human health hazard as well as an animal welfare problem. Furthermore, the economic implications in terms of trade restrictions and productivity losses have direct and indirect implications for human health and the food supply.

Studies of mice, humans, and cattle have shown that antigen-specific CD4+, CD8+, and γ/δ T cells are activated following exposure to mycobacteria or derived antigens (12, 15, 27, 31, 39). CD4+, CD8+, and γ/δ T cells are recruited to the site of infection and are capable of producing gamma interferon (IFN-γ) and tumor necrosis factor alpha (9).

Murine studies, including depletion with monoclonal antibody (MAb), adoptive transfer, and gene disruption, have shown the critical involvement of CD4+ and CD8+ T cells in controlling infection (12). The role of γ/δ T cells in immunity to mycobacteria is less clear, as the susceptibility of δ T-cell receptor-deficient mice appears to be dependent on the dose and strain of mycobacterium used for infection (23, 24). In cattle, depletion studies with MAbs have provided evidence for the involvement of WC1+ (γ/δ) cells in the immune response to M. bovis (36).

Immunity mediated by T cells can function in a number of ways, including contribution to the production of cytokines, notably IFN-γ, tumor necrosis factor alpha, and lymphotoxin α, that are central to macrophage activation and granuloma formation (8, 19). T cells are also able to kill mycobacterium-infected cells (32, 33, 37, 48). The killing of infected cells can result in either the release of intracellular bacteria or killing of both the infected cell and the infecting bacteria. It has been shown that ATP can induce apoptosis of macrophages infected with mycobacteria, as well as inducing the killing of the infecting pathogen (25, 50), and it is postulated that secretion of extracellular ATP directed to the infected macrophage could be a mechanism by which T cells stimulate the killing of intracellular mycobacteria (50). More recently, CD8+ T cells have been shown to release granulysin into infected macrophages following the delivery of perforin, which would kill the host cell and then kill the infecting mycobacteria; granulysin has been shown to be capable of killing free-living mycobacteria (48). Thus, CD4+ and CD8+ cells contribute to the formation of the TB granuloma and the arrest of mycobacterial growth mainly by the expression of a T helper type 1 (Th1) response.

Although a role for T cells in immunity to mycobacteria has been shown directly (in vivo) in mice and indirectly (in vitro) in mice, humans, and cattle, there are few studies documenting the direct (in vivo) involvement of T cells in immunity to mycobacteria in other species. Here, we report that in vivo depletion of CD8+ T cells in cattle in the early stages of infection with M. bovis did not affect the ability of peripheral blood cells to proliferate in response to mycobacterial antigens but did reduce their ability to produce IFN-γ. Depletion also resulted in a lower level of pathology in the head lymph nodes than for nondepleted control calves. These results suggest that CD8+ cells play a role in the immunopathology of TB.

MATERIALS AND METHODS

Cattle.

Ten male Friesian calves 2 to 4 weeks of age obtained from a TB-free farm were divided into two groups of five. The calves were housed in appropriate containment category III facilities for 4 weeks prior to any experimentation. This period was used to observe the calves and to ensure their freedom from preexisting respiratory infections. All experiments conformed to local and national guidelines on the use of experimental animals and category III infectious organisms.

Inoculation of cattle with M. bovis and depletion of CD8+ T cells.

The cattle were inoculated intratracheally with 104 CFU of M. bovis strain AF2122/97. Pilot experiments indicated that this strain at this dose and by this route induced TB in calves 6 to 8 weeks of age. The inoculum titer was confirmed by colony counts on 7H10 agar plates. Ten days after inoculation with M. bovis, the calves were injected intravenously with 10 mg of the CD8-depleting MAb CC63 (52) (CD8-depleted calves) or 10 mg of the isotype control MAb AV55 directed against a chicken leukocyte antigen (F. Davison, personal communication) (control calves) for seven consecutive days. In pilot experiments, this protocol proved to reduce the number of CD8+ T cells in peripheral blood (data not shown). Additionally, MAb CC63 has been shown to affect the course of respiratory syncytial virus infection, indicating that it is capable of depleting CD8+ T cells in the respiratory tract (52). Prior to the inoculation of MAb, the calves were injected with flunixin meglumine (Schering and Plough, Uxbridge, Middlesex, United Kingdom) to prevent the occurrence of an anaphylaxis-like reaction due to the depletion of CD8+ T cells. The depletion of CD8+ T cells was monitored in peripheral blood cells by flow cytometry as described below.

Measurement of immunological responses to M. bovis antigens.

Blood was collected in heparin (final concentration, 10 IU/ml) to monitor immune responses. IFN-γ was measured in plasma taken from blood incubated with RPMI 1640 medium or purified protein derivative (PPD) from Mycobacterium avium (PPD-A) or M. bovis (PPD-B) (VLA, Surrey, United Kingdom) at 20-μg/ml final concentration at 37°C in an atmosphere of 5% CO2 and 95% humidity for 24 h. This protocol is similar to that used to determine IFN-γ production against mycobacterial antigens for diagnostic purposes (57). The blood was centrifuged at 800 × g for 10 min, and the plasma was harvested and stored at −20°C until it was assayed. The concentration of IFN-γ was determined by standard capture enzyme-linked immunosorbent assay as described previously (22) using MAb CC330 as the capture antibody and MAb CC302 as the detection antibody. To determine proliferative responses, 200 μl of blood/well diluted 1:10 with RPMI 1640 containing glutamax-II (Gibco, Paysley, United Kingdom) was incubated in triplicate in 96-well round-bottom plates for 6 days with medium alone or with 10 μg of PPD-A or PPD-B/ml (final concentration). For the last 18 h, 37 mBq of [3H]thymidine (3H-TdR) (Amersham International, Amersham, United Kingdom)/well was added, and the plates were frozen. The plates were thawed, paraformaldehyde was added to a final concentration of 1%, and the plates were incubated for 1 h at room temperature to eliminate the risk of live mycobacteria being present in the sample. The cells were harvested with a Skatron semiautomated cell harvester onto glass fiber filter mats (Walac, Truku, Finland). The incorporated radioactivity was determined in a scintillation counter (Pharmacia, Uppsala, Sweden) as counts per minute. The results are expressed as stimulation index (SI), which is the counts incorporated by the cells cultured in the presence of antigen divided by the counts incorporated by the cells cultured in medium alone.

Flow cytometry.

To monitor peripheral blood cell populations, blood was placed in tubes with 5 volumes of Gey's solution (29) containing 1% paraformaldehyde. After 1 h of incubation at room temperature, the peripheral blood leukocytes (PBL) were pelleted at 500 × g for 10 min and washed three times with phosphate-buffered saline (PBS) by centrifugation at 400 × g for 5 min. Phenotypic analysis of the cells was carried out as described elsewhere (17) with slight modifications. In brief, 106 cells were incubated with MAb directed against bovine leukocyte antigens for 10 min at room temperature. The cells were washed three times in PBS, incubated with goat antibodies specific for mouse isotype immunoglobulin G1 (IgG1), IgG2a, or IgG2b (Southern Biotechnology Associates) for 10 min, washed as described above, and analyzed in a FACScalibur (Becton Dickinson). Staining was performed using MAbs to bovine CD4 (CC8), CD8 (CC63), WC1 (CC15), CD14 (CCG33), major histocompatibility complex class II (IL-A88), and CD25 (IL-A111), together with control MAbs AV20, AV29 (42, 43), and AV37 (F. Davison, personal communication) directed against chicken antigens. The data are expressed as percentages of target cells in the total PBL.

Postmortem examinations.

The following tissues were examined at postmortem: head lymph nodes (parotid, submandibular, and retropharyngeal), lower respiratory tract-associated lymph nodes (mediastinal and up to four bronchial nodes), tonsils, and lungs. The tissues were sliced at 0.5- to 1-cm intervals and examined macroscopically. To determine the magnitude of the lesion, the following scoring system was used (55). Lymph nodes were assigned 0 for no visible lesion, 1 for a small focus 1 to 2 mm in diameter, 2 for several small foci 1 to 2 mm in diameter or a necrotic area of 5 by 5 mm, and 3 for extensive necrosis. Lungs were assigned a value of 0 for no visible lesions, 1 for no gross lesion but lesions apparent upon slicing, 2 for up to five lesions <10 mm in diameter, 3 for more than six lesions <10 mm in diameter, 4 for one distinct lesion >10 mm in diameter, and 5 for gross coalescing lesions.

Bacterial counts in organs.

Organ biopsy specimens of ~1 cm3 (1 g) were macerated using sand, pestle, and mortar in PBS. Serial dilutions were plated on modified 7H11 agar plates (55) and incubated at 37°C. After 4 weeks, colonies were counted; the results are expressed as log10 CFU per gram of tissue. This period of incubation with this strain has proven to be enough to determine whether the cultures are negative.

Histology.

Tissue biopsy specimens were fixed in 10% neutral buffered formalin for at least 7 days before being processed. The biopsy specimens were embedded in wax, and 4-μm-thick sections were cut and stained with hematoxylin and eosin using standard procedures.

RESULTS

MAb CC63 transiently depletes CD8+ T cells in cattle.

Injection of MAb CC63 has been shown to deplete CD8+ T cells in peripheral blood and in the lungs and to influence the progress of an experimental respiratory syncytial virus infection in cattle (52). We confirmed that the regime followed in the present experiments was capable of temporarily depleting the CD8+-T-cell population in a pilot experiment with uninfected calves (data not shown). Injection of MAb CC63 into calves infected with M. bovis induced, as expected, a marked transient depletion of CD8+ T cells in peripheral blood (Fig. (Fig.1),1), which was statistically significant at week 2 (P = 0.01 by the t test) and occurred in all five inoculated calves. Shortly after antibody treatment was stopped, the CD8+ T cells reappeared.

FIG. 1.
(A) Number of PBL per milliliter in peripheral blood. (B to D) Average percentages of PBL stained with MAb in cattle infected with M. bovis. The cells were stained for CD4+ (B), CD8+ (C), and WC1+ (D) cells as described in Materials ...

Proliferative responses of 1:10-diluted blood to mycobacteria antigens.

3H-TdR incorporation in response to PPD-B was detectable in all animals from both groups by week 4 postinfection (Fig. (Fig.2).2). Some variability was noted throughout the experiment; however, the SI never dropped below 10 for any of the animals in either group.3H-TdR incorporation in response to PPD-A (not shown) was detectable in some animals of both groups at different times, but it was much lower than that seen with PPD-B. No differences were detected in the proliferative responses of the depleted and nondepleted groups.

FIG. 2.
Proliferative responses of PBL from individual control (A) and CD8+-T-cell-depleted (B) calves to PPD-B expressed as SI. The continuous line represents the median SI of the group, and the number next to each bar is its numeric value of the average. ...

Production of IFN-γ in blood in response to mycobacterial antigens in vitro.

Production of IFN-γ against PPD-A (not shown) or PPD-B (Fig. (Fig.3)3) was evaluated in the plasma of 24-h whole-blood cultures. The production of IFN-γ in response to medium alone remained undetectable throughout the experiment, and therefore it is not shown. IFN-γ against PPD-B was detected in some animals in the two groups by 3 weeks after inoculation and in all animals in both groups by 4 weeks after inoculation and remained relatively high throughout the experiment. A comparison of the production of IFN-γ against PPD-B in the two groups from week 4 onward shows that control calves produced more IFN-γ than depleted calves (P = 0.0367 by a Mann-Whitney test). At the peak of the response at week 5, control calves produced IFN-γ with a range from 40.7 to 469.8 pg/ml, while depleted calves in the same week produced IFN-γ with a range of 55.3 to 117.9 pg/ml. Thus, depletion of CD8+ T cells early during infection appeared to compromise the ability of the animals to mount high IFN-γ responses to mycobacterial antigens later in infection. IFN-γ production against PPD-A (not shown) was detectable by week 4 postinfection and reached a peak at 4 to 7 weeks postinfection, with a median production of 6.25 pg/ml for the control group and 4.18 pg/ml for the CD8-depleted group. However, the production of IFN-γ against PPD-A compared to that in response to PPD-B was minor. No correlation was found between proliferative responses and production of IFN-γ in response to PPD-B.

FIG. 3.
IFN-γ production in picograms per milliliter by PBL from individual control (A) and CD8-depleted (B) calves against PPD-B. The continuous line represents the median of the group, and the number next to each bar is its numeric value. Each symbol ...

Evaluation of TB lesions at postmortem.

Table Table11 shows the outcome of the evaluation of the presence of TB lesions at postmortem, 8 weeks after challenge. No differences were detected between the two groups in the levels of lesions in the lungs and associated lymph nodes. This is reflected in the lesion scores for these organs, with a cumulative score of 66 for calves depleted of CD8+ T cells and 69 for control calves. However, the score for TB lesions in the head lymph nodes showed a difference between the two groups: CD8+-T-cell-depleted animals had a total score of 21 in these tissues. On the other hand, the control calves had a score of 53 in these tissues. Although not statistically significant (P = 0.115), these differences show a trend indicating that CD8 cells contribute to pathology.

TABLE 1.
Lesions in lymph nodes of control or CD8-depleted calves at postmortem, 8 weeks after inoculation

The studies described indicated a possible relationship between production of IFN-γ and pathology. The amounts of IFN-γ produced by PBL in response to PPD-B in vitro from each calf throughout the experiment were added, and the total was plotted against the pathology score. The plot (Fig. (Fig.4)4) shows a direct correlation between production of IFN-γ and gross pathology. Thus, it would appear that a general effect of depleting CD8 cells is diminished production, but not total absence, of IFN-γ, which is associated with diminished pathology.

FIG. 4.
Correlation between cumulative production of IFN-γ by whole blood cells against PPD-B and gross pathology score (P ≤ 0.001; R2 = 0.7898). Squares, control calves; crosses, depleted calves.

Mycobacterial viable counts in lymph nodes and histological examination.

Table Table22 shows the results of the culture of selected lymph node biopsy specimens on 7H11 agar. The limit of detection of the technique employed is five bacteria. Mycobacteria were isolated from mediastinal lymph nodes (Table (Table2),2), but not from head lymph nodes (retropharyngeal or parotid). No differences were detected between depleted and control calves. Although no bacteria were detected in parotid or retropharyngeal nodes, upon histological examination, it was confirmed that the lesions were typical of M. bovis, showing giant Langhan's cells within developing areas of necrosis, and in more advanced lesions, calcification was evident. Thus, the depletion of CD8 cells early after infection did not have consequences for the bacterial load at postmortem, 8 weeks after infection.

TABLE 2.
Mycobacterial viable counts in lymph nodes of control or CD8-depleted calves at postmortem (8 weeks after inoculation) expressed as log10 CFUa

DISCUSSION

In this work, we evaluated the role of CD8+ T cells in immunity to M. bovis in cattle in vivo by depleting target cells with MAb. PBL from animals depleted of CD8 cells produced reduced amounts of IFN-γ in vitro in response to M. bovis antigens, indicating that in vivo these cells play a role in the immune response to mycobacteria by contributing to the amount of IFN-γ produced postchallenge. However, CD8 cells also appeared to contribute to the immunopathology of bovine TB, possibly through the regulation, or the production, of IFN-γ.

The importance of CD8 cells in protection against TB in vivo was first shown in adoptive-transfer experiments with mice (35, 36). Later experiments, using depletion of CD8 cells by MAb in mice infected with M. bovis, further showed that these cells play a role in immunity to mycobacteria (30). More recently, mice genetically deficient in β2m or TAP have been used to demonstrate a role for major histocompatibility complex class I-restricted cells in immunity to tuberculosis (13). Further evidence comes from immunization studies with dendritic cells pulsed with CD8 cell-restricted epitopes that were found to be protective (28). In humans, a role for CD8 T cells in immunity is suggested by studies showing the presence of specific immune cells after infection or vaccination with M. bovis BCG (4, 45-47, 49). In cattle, like humans, a role for CD8 cells can be inferred from the presence of antigen-specific immune CD8 T cells following infection with M. bovis or vaccination with BCG (15, 26, 27).

To study the role of CD8 cells in immunity to mycobacteria in cattle, we used MAb to deplete the target population. However, depletion using MAb can only be carried out for a limited time, as an anaphylactic reaction to murine IgG develops in calves ~10 days after initial inoculation (16). Therefore, any effects seen in this experiment would be the result of the temporary depletion of the target population while MAb was being inoculated. We have previously used similar depletion protocols to target T-cell subpopulations for depletion in cattle to investigate their roles in different infections. The clearest findings have been with viruses causing acute transient disease, and they have clearly shown the involvement of CD4 and CD8 cells in immunity to infection (16, 34, 52). Using a similar protocol, depletion of WC1+ cells in cattle infected with M. bovis provided evidence that these cells contributed to the IFN-γ response in vivo and to the Th1 bias seen in bovine TB (21).

We chose to start CD8 cell depletion 10 days after inoculation with M. bovis, as in our experience and that of others, specific immune responses to mycobacteria are detected 2 to 4 weeks after infection (3, 39, 40, 55). Furthermore, it has been reported that CD8 cells appear early, after 1 to 2 weeks, in response to infection with mycobacteria in mice (44). Thus, the timing of the inoculation of MAb should have coincided with the early stage of the immune response at the site of infection, ensuring that the CD8 response would be delayed and that any effect would be magnified over the ensuing period. Measurement of the level of CD8 cells in peripheral blood indicated that, indeed, this population was temporarily depleted while MAb was being inoculated and recovered quickly after administration of MAb ceased. This is in agreement with previous work showing that CD8 depletion is difficult to maintain (16, 52).

The mechanisms by which CD8 cells contribute to immunity to mycobacteria include the production of cytokines and direct lysis of infected cells (for a recent review, see reference 12). An important mechanism by which CD8 cells contribute to protection against mycobacteria is the production of IFN-γ. CD8 cells have been shown to produce IFN-γ in response to mycobacteria in mice, humans, and cattle (7), thereby contributing to the activation of macrophages and the formation of granulomas for the containment of mycobacteria. In mice, production of IFN-γ has been shown to be essential for CD8-mediated protection (51). IFN-γ has been shown to be an essential cytokine in immunity to mycobacteria. Mice or humans deficient in any component of the IFN-γ cascade have been shown to be more susceptible to mycobacteria than their wild-type counterparts (18). In mice, it has been demonstrated that one mechanism by which IFN-γ contributes to protection is by activating macrophages to kill intracellular mycobacteria (1, 10). However, in humans and cattle, the role of IFN-γ in the activation of macrophages for the killing of mycobacteria has been more difficult to pinpoint (2, 53), as preincubation of human or bovine macrophages with IFN-γ alone does not induce the activation of macrophages to kill intracellular mycobacteria in vitro. In the human and bovine systems, the presence of lymphocytes is required for the control of mycobacteria, although in the bovine system, this phenomenon is not antigen specific (5). In this work, we found no differences between CD8-depleted and control groups in the incorporation of 3H-TdR by PBL in response to PPD-A or PPD-B. On the other hand, PBL from CD8-depleted calves produced less IFN-γ in response to mycobacteria than PBL from control calves. Thus, depletion of CD8 cells in vivo resulted in a diminished ability of PBL to produce IFN-γ in response to mycobacterial antigens in vitro. It is unlikely that lower production of IFN-γ was due to a lower percentage of CD8+ cells in peripheral blood at the time the in vitro test was carried out. From week 4 onward, when the differences between CD8-depleted and control calves became apparent, the CD8+ population in the peripheral blood of depleted calves had recovered from the trough of depletion at week 2. Further, PPD-B would have been loaded into the exogenous antigen presentation pathway and therefore would have been unlikely to stimulate CD8+ cells during a 24-h incubation. Rather, it is likely that through the production of IFN-γ in vivo, CD8+ T cells contribute to the Th1 polarization of the immune response, the results of which are reflected in the in vitro production of IFN-γ against mycobacterial antigens, mainly by CD4+ T cells (reference 56 and our unpublished observations).

Due to their reduced ability to produce IFN-γ in response to PPD-B in vitro, it would have been expected that CD8-depleted calves would be more susceptible to infection than controls. Postmortem examination revealed that depleted and control calves had similar levels of pathology in the lower respiratory tract lymph nodes, but CD8-depleted calves had a lower lesion score in the head lymph nodes than control calves. Further analysis of these results also revealed a positive correlation between production of IFN-γ and the gross pathology score, indicating that IFN-γ could be involved in the pathogenesis induced by M. bovis.

It might have been expected that a reduced ability to produce IFN-γ in response to mycobacterial antigens in CD8-depleted calves would have consequences for the bacterial load in these calves. On the other hand, the number of lesions might be expected to be related to the bacterial load and therefore to be greater in control calves. No indication of differences in the numbers of mycobacteria detected in depleted or control lymph nodes was found, although histological examination revealed lesions typical of TB. It is necessary to interpret bacterial counts with caution; for instance, it has been shown that the level of pathology in cattle is disproportionate to the bacterial load compared to the pathology levels and bacterial loads seen in other animal species (14). Also, to determine the bacterial load, we used 1 g of tissue, which may not be fully representative.

Thus, although it has been shown that CD8+ cells are required for protection against mycobacteria, it is possible that through regulation of the production of IFN-γ, CD8 cells may also contribute to pathogenesis in the bovine model of TB. This would also imply that, like CD8+ cells, IFN-γ, although necessary for protection, also contributes to the pathology of M. bovis in cattle. It is possible that following CD8 depletion in the early stages of infection, an altered balance of the immune response occurred from increased CD4 cell contact with infected antigen-presenting cells, which permitted the establishment of a more protective immune response. By the time CD8 cells returned to normal levels, their response would have been directed by the already established immune response toward protection rather than pathology. Although production of IFN-γ is regarded as necessary for protection, it has been shown in mice to be involved in the immunopathology induced by M. avium (11) and respiratory syncytial virus (38). In humans, the levels of IFN-γ in the serum or pleural fluid have been shown to be positively correlated with the level of disease (41, 54, 58). Recently, it has been proposed that the induction of a strong unregulated Th1 response may be a strategy of mycobacteria for survival in the host. The formation of the granuloma, followed by its liquefaction, may provide a rich environment to which the immune system has no access and in which the mycobacteria can replicate extracellularly (20). Immunity against mycobacteria is multifactorial and dependent on the balance between an inflammatory response that allows the host to develop a granuloma, which contains the microorganism, and an anti-inflammatory response that restricts the extent of the granuloma and allows contact of effector T cells with the infected cells, which results in the killing of the infecting pathogen. While a Th1 response is necessary for protection, it may also have immunopathological consequences.

Acknowledgments

We thank M. Vordermeier and G. Hewinson for providing the virulent M. bovis AF2122/97 strain. We also acknowledge the contribution of staff in the HSU.

This work was supported by grants from the BBSRC and DEFRA.

Notes

Editor: S. H. E. Kaufmann

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