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Antigen Recognition and Immunomodulation by T Cells in Bovine Tuberculosis¹

TB Research Group, Veterinary Laboratories Agency, Surrey, United Kingdom

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
This report describes the in vitro proliferative responses of peripheral blood T cells to defined mycobacterial protein Ags and the immunomodulatory effect of T cells in cattle infected with Mycobacterium bovis. T cell responses were specific to M. bovis infection because they were detected in cattle either experimentally or naturally infected with M. bovis, but were not present in uninfected controls. Proliferating T cell cultures produced enhanced levels of IFN- and TGF-, but not IL-2 in response to the more immunodominant mycobacterial Ags. Depletion of T cells from PBMC resulted in an increased Ag-specific proliferation in half the animals tested, indicating a suppressive effect of T cells upon other () T cell responses. Because T cells constitute a major T cell population in the peripheral blood of cattle, the activities of T cells described in this report could make a significant contribution to the immune response in bovine tuberculosis.

	Introduction

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Tuberculosis is currently increasing in the British herd. As a result of this, the need for new vaccines and diagnostic reagents was highlighted in a recent independent scientific review (1). For the rational development of such reagents it is useful to understand something of the underlying immune mechanisms that constitute this immunopathological disease. In this report we concentrate upon the activity of the T cell and speculate upon its role in bovine tuberculosis.

T cells were identified >15 years ago (2) and yet the physiological role of these cells and their ligands is still not fully understood. They have been shown to accumulate in the infected tissues both in mice (3) and humans (4) infected with M. tuberculosis. In mice, there are conflicting reports as to whether T cells offer protective immunity (5), or not (6) against M. tuberculosis and M. bovis bacillus Calmette-Guérin (BCG),³ respectively. Other reports identify a possible role for T cells in cellular trafficking following infection with M. tuberculosis (7), or in the early establishment of the cytokine profile following infection by a pathogen (8). In humans T cells have been shown to be increased in the blood of anergic, skin-test-negative tuberculosis patients (9), suggesting a suppressive role of T cells in these individuals.

Very few ligands for T cells have been identified (10). Well-defined mycobacterial ligands for T cells include the heat shock protein (hsp) 65 in both mice and humans (11, 12), and nonpeptide Ags in humans (13). Other reports have described reactivity to as yet unknown low molecular weight protein Ags of M. tuberculosis in humans (14, 15).

Cattle and other ruminants, unlike humans and mice (16, 17), have large numbers of T cells in the peripheral blood (18, 19, 20). At least two distinct subpopulations of T cells have been identified based on the mutually exclusive expression of the surface markers WC1, CD2, and CD8, and these subpopulations are uniquely distributed in various tissues (21). In bovine peripheral blood the majority of T cells express WC1, with the phenotype WC1⁺CD2^-CD8^-, while a small population of T cells are WC1-negative, but express CD2 and, largely, CD8 (WC1^-CD2⁺CD8^+/-) (22). Bovine peripheral blood T cells have been shown to proliferate in response to Theileria-infected cells in the presence of IL-2 (23) and down-regulate T cell responses in bovine Fasciola infection (24). T cell lines have also been demonstrated to be effective Ag-presenting cells for CD4⁺ T cells, causing their proliferation (25).

In this investigation of bovine tuberculosis, we have asked two questions: 1) do bovine peripheral blood T cells respond to mycobacterial protein Ags; and 2) what effect, if any, do T cells have on the activity of other T cells. This report shows that bovine T cells can both proliferate and enhance cytokine production in response to protein Ags, and describes an immunoregulatory function of T cells that can suppress Ag-specific responses of other () T cells.

	Materials and Methods

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Animals

Six female friesian Limousin cross calves experimentally infected with M. bovis (CP1662, CP1665, CP1759, CP1760, CP1824, CP1825, for methods see below) and one naturally infected M. bovis field reactor (CK556) were used for this investigation. Four uninfected animals were used as controls where indicated. Experimental cattle, 6 months of age were obtained from a herd free of bovine tuberculosis (i.e., with a history of negative skin test results). The calves were inoculated intratracheally according to the protocol previously described (26, 27) with 2.5 x 10⁴ CFU M. bovis (AF2122/97). During the study, animals were housed in a high security isolation unit under negative pressure, and expelled air was filtered. Two weeks prior to postmortem all animals were tested using the single comparative intradermal tuberculin skin test (SCITT) using avian and bovine tuberculins according to the European Economic Community directive (28). These cattle all gave a positive SCITT test. A detailed postmortem of the experimentally infected animals showed tuberculous lesions in the upper lungs and respiratory lymph nodes in all animals, identified acid-fast bacilli in lesioned tissue by histopathology, and cultured M. bovis from lesioned material (data not shown). Blood was collected from experimentally infected animals prior to postmortem, between 16 and 20 wk after infection with M. bovis.

Mycobacterial Ags

Bovine and avian tuberculin preparations (PPD-M and PPD-A; to assess exposure to M. bovis and M. avium respectively, M. avium being representative of a widely distributed environmental mycobacteria), and recombinant proteins ESAT-6, MPB70, and MPB83 and the ESAT-6 peptides were produced at the Veterinary Laboratories Agency (VLA) as previously described (29). Recombinant MPB64 was provided by D. Bakker (Animal Health Science, Boxtel, The Netherlands). Native Antigen 85, isolated from BCG culture filtrate and comprised of Ag85A, B, and C, was donated by K. Huygen (Instituut Pasteur, Brussels, Belgium). Recombinant hsp16.1, hsp65, and hsp70, and the 38 kDa lipoprotein were a gift from M. Singh (GBF, Braunschweig, Germany). Details of these mycobacterial Ags are given in Table 1.

PBMC were separated from heparinized venous blood over histopaque 1077 (Sigma, Poole, Dorset, U.K.) and resuspended in culture medium (RPMI 1640 with Glutamax (Gibco, Life Technologies, Paisley, U.K.) supplemented with 5% CPSR (serum replacement, Gibco), nonessential amino acids (Life Technologies), 100 U/ml penicillin, 100 µg/ml streptomycin, and 5 x 10^-5 M 2-ME (Gibco). Cells were resuspended in culture medium or were used in further T cell sorting and staining procedures (see below).

Magnetic T cell sorting

T cell subsets were isolated or depleted from PBMC using the MidiMACS magnetic sorting system (Miltenyi Biotech, Bisley, Surrey, U.K.). T cells were labeled using the mAb GB21A (31) (IgG2b; VMRD, Pullman, WA). Ab-labeled PBMC were then incubated with goat anti-mouse IgG microbeads (Miltenyi Biotech) before positive sorting on magnetic columns. TCR⁺ T cells thus prepared were >97% pure as determined by flow cytometry following column separation. Removal of T cells from PBMC was equally efficient. Positively sorted TCR⁺ T cells or -depleted PBMC were then washed and resuspended in culture medium.

Proliferation assays

PBMC and -depleted PBMC. Cells (2 x 10⁵) were added per well to 96-well flat-bottomed microtiter plates (Nunc, Life Technologies, Paisley, U.K.). Ags were added in triplicate (10 µg/ml final concentration for all Ags used in the study).

TCR⁺ T cells. T cells (2 x 10⁵) were added per well to 96-well flat-bottomed microtiter plates (Nunc) with 2 x 10⁵ mitomycin C-treated PBMC as feeder cells (40 µg/ml mitomycin C in PBS for 30 min at 37°C, 5% CO₂). Ags were added in triplicate to a final concentration of 10 µg/ml. Peptides were added to a final concentration of 25 µg/ml. Control feeder cell-only (plus Ag) cultures were set up at the same time. Parallel /feeder and feeder-only cultures were also set up to collect supernatants for the measurement of cytokines and to assess the cell populations of responding cultures by flow cytometry.

Cultures for proliferation were incubated for 5 days at 37°C in 5% CO₂, pulsed with 37 kBq tritiated thymidine/well (Amersham, Amersham, U.K.), and harvested 24 h later. Incorporated radioactivity was determined as cpm by -scintillation counting. A positive result (Ag-specific proliferation) was identified using 99% confidence intervals, i.e., the mean cpm in the presence of Ag to be greater than the mean cpm of control-unstimulated cultures plus 9.925 x SEM of control cultures (32). Supernatants and cells for the measurement of cytokines and flow cytometry, respectively, were harvested on the 6th day of culture. Supernatants were stored at -20°C. Cells were immediately processed (see below).

Flow cytometry

PBMC and T cell cultures were analyzed using the mAbs GB21A (IgG2b, TCR; (31), CC-30 (IgG1, CD4; (33), CC-58 (IgG1, CD8; (34), and CC-15 (IgG1, WC1; (35) (CC-30, CC-58, and CC-15 courtesy of IAH, Compton, Berkshire, U.K.). FITC- or RPE-conjugated goat anti-mouse isotype-specific secondary Abs (Serotec, Oxford, U.K.) were used for the double-labeling of cells. Cells (10⁶) were washed in PBS containing 0.1% sodium azide before resuspending in primary Ab (culture supernatants diluted 1:10, GB21A at 5 µg/ml) for 10 min at room temperature. The cells were washed in PBS/azide and resuspended in the secondary conjugated Abs (diluted according to manufacturer’s instructions) for 10 min at room temperature. After washing again the cells were resuspended in 1% paraformaldehyde in PBS and stored at 4°C until analyzed using a FACScan and CellQuest software (Becton Dickinson).

Cytokine assays

IFN- ELISA. Neat culture supernatants were assessed for IFN- content using a commercially available Ag capture ELISA kit, BOVIGAM (CSL, Parkville, Victoria, Australia) and following the manufacturer’s instructions. Results are expressed as the mean optical density (O.D. 450 nm) of duplicate supernatants plus the SD.

IL-2 bioassay. IL-2 activity was measured by the ability of each culture supernatant to stimulate Con A lymphoblasts, following the method of Emery et al. (36). Neat culture supernatants (50 µl) were added to the Con A blasts (10⁴/well) in duplicates. Serial dilutions of recombinant human IL-2 (R&D Systems Europe, Abingdon, U.K.) provided the standard curve. The plates were incubated for 24 h at 37°C in 5% CO₂, pulsed with tritiated thymidine, and harvested 24 h later. The specificity of this bioassay for IL-2 has been previously determined by inhibition with a mAb specific for the subunit of the IL-2 receptor CD25 (37).

TGF- bioassay. Culture supernatants were tested for the presence of TGF- using Mv1Lu cells. Mv1Lu cells were plated out in 96-well flat-bottomed microtiter plates at 2 x 10⁴ cells/well in 0.1 ml IMDM (Gibco) supplemented with 2% FCS and incubated overnight at 37°C, 5% CO₂. Serial dilutions of recombinant human TGF- (R&D Systems Europe) provided the standard curve. Fifty microliters of standard dilutions or neat samples were added to the Mv1Lu cells in duplicate, and plates were incubated overnight. Plates were then pulsed with tritiated thymidine, incubated for another 24 h, and then freeze-thawed before harvesting.

	Results

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T cells proliferate in response to mycobacterial protein Ags.

The proliferation of purified T cells to a panel of mycobacterial Ags was measured in seven animals (six experimentally infected with M. bovis, and one naturally exposed to M. bovis). Fig. 1 shows the T cell-proliferative responses in three representative animals (CP1760, CP1824, CP1825) experimentally infected with M. bovis. Positive responses (using 99% confidence limits) compared to respective medium/no Ag controls are indicated by asterisks. Ags that caused the most prominent responses in all seven cattle tested were PPD-M, Ag85, and ESAT-6. Other notable T cell responses were observed to the Ags MPB83 and hsp16.1 in 6/7 animals. One experimentally infected animal (CP1825), shown in Fig. 1, also responded strongly to MPB70. The naturally infected field reactor animal, CK556 (data not shown), responded to a broader range of Ags (including MPB70, MPB64, and hsp70) compared with experimentally infected animals, and responses of this animal were also relatively stronger, possibly owing to the longer term infection of this animal. T cells were also found to proliferate in response to peptides of ESAT-6 (data not shown). Interestingly, although some peptides elicited strong T cell and PBMC responses, others induced only weak T cell proliferation in the face of strong PBMC activity. This further supports T cell responses as being independent, rather than reflective, of the responses of other cells. In contrast to the responses observed above, none of these animals responded to the nonpeptide Ags TUBag1, IPP, or Epox (courtesy of J. J. Fournie, Centre Hospitalier Universitaire Purpan, Toulouse, France; data not shown).

View larger version (18K): [in this window] [in a new window]   FIGURE 1. Proliferation of purified T cells to a panel of mycobacterial antigens. T cells were incubated at a ratio of 1:1 with mitomycin C-treated autologous PBMC feeder cells plus 10 µg/ml of each Ag, or medium as a control. Results are expressed as cpm of triplicate cultures (i.e., the mean response of -plus-feeder cell cultures minus the mean response of feeder cell-only control cultures) for each Ag and for the medium control. *, Positive response of T cells to Ag compared with the medium (no Ag) control for each animal (99% confidence limits).

View larger version (27K): [in this window] [in a new window]   FIGURE 2. No T cell proliferation was observed in the control uninfected cattle. Two of four animals (CP1754, CP1755) showed substantial and equivalent PBMC proliferation to both PPD-A and PPD-M tuberculin preparations thus indicating exposure to environmental mycobacteria, but not exposure to M. bovis. However, even in these animals there was no T cell response to tuberculin. The response of T cells to the mitogen Con A shows that the purified T cells were viable. Data show the mean proliferation (cpm) of triplicate cultures plus the SEM.

View larger version (24K): [in this window] [in a new window]   FIGURE 3. Flow cytometric analysis of responding T cell cultures for one representative animal, CP1824. Cell populations within responding/proliferating T cell-plus feeder cultures were analyzed following stimulation with Ag, or medium as a control, for 6 days. a, Scatter plots for one animal following stimulation with either ESAT-6 or medium and illustrates the increase in Region 2 (R2) cells following specific stimulation. b, Positive staining of R2 cells with the TCR-specific Ab GB21A (solid green) against an isotype-matched control Ab. c, Positive correlation between the proliferation of purified T cells and the number of cells in R2 as a proportion of total cells in response to each Ag. Each dot represents one Ag.

Enhanced Ag-specific IFN- and TGF-, but not IL-2, by T cells

Cytokines were measured in those culture supernatants where T cell proliferation was observed, and also in the no-Ag, unstimulated (medium) control T cell culture supernatants as a control for spontaneous release. Enhanced IFN- production by T cell cultures (compared to feeder-only cultures) was observed in response to PPD-M in 6/6 animals, to Ag85 in 4/6 animals, to ESAT-6 in 4/6 animals, and also to hsp16.1 in 1/3 animals tested. Enhanced TGF- production in T cell cultures (compared with feeder-only cultures) was observed in response to PPD-M in 4/6 animals, ESAT-6 in 5/6 animals, Ag85 in 3/6 animals, MPB83 in 3/4 animals tested, and hsp16.1 in 2/4 animals tested. There was also some enhancement of TGF- activity of T cell cultures in the absence of any Ag stimulation (medium control) in 5/6 animals. Fig. 4 shows the levels of IFN- (Fig. 4a) and TGF- (Fig. 4b) measured in Ag- or medium-stimulated T cell cultures ( plus feeder) compared with feeder-only cultures in three representative animals (CP1665, CP1759, CP1760). IL-2 activity was barely detectable in either the T cell or feeder-only culture supernatants. Both 6-day and 24-h supernatants were assayed for IL-2, and these were all <0.6 and <1 U/ml, respectively (data not shown). Furthermore, no differences in these low levels of IL-2 were observed between supernatants from Ag-stimulated or control unstimulated T cell cultures.

View larger version (26K): [in this window] [in a new window]   FIGURE 4. IFN- (a) and TGF- (b) responses of proliferating -plus-feeder () compared to feeder-only () cultures for three representative animals, CP1665, CP1759, and CP1760. IFN- responses are expressed as the mean OD450 nm plus SD of duplicate cultures. TGF- responses are expressed as ng/ml in neat supernatants.

Immunomodulation of T cell proliferation by T cells

The effect of T cell depletion on PBMC responses to mycobacterial Ags was measured in six animals. The removal of T cells from PBMC increased the Ag-specific proliferation in 3/6 animals tested (CK556, CP1662, CP1825) (Fig. 5). Not only did T cell depletion enhance existing responses to various Ags, but in some cases where there was no detectable PBMC response to Ag, e.g., all Ags shown for CP1662 in Fig. 5, and Ags hsp65 and 38 kDa in CP1825, the removal of T cells revealed strong proliferative responses in the remaining T cell population. This suggests that specific T cell clones were present in PBMC from these animals but were being suppressed by the T cells. T cell depletion had a less profound effect upon PBMC responses to those Ags that induced very strong proliferative responses, i.e., PPD-M, ESAT-6, Ag85, hence these Ags are not included in Fig. 5. Background PBMC proliferation following T cell depletion was increased only slightly (CK556, CP1662) to moderately (CP1825) in these animals. Positive proliferation responses of -depleted PBMC to the individual Ags, compared to the no-Ag medium controls (using 99% confidence limits) are indicated by asterisks.

View larger version (25K): [in this window] [in a new window]   FIGURE 5. Increased Ag-specific responses of T cell-depleted PBMC. Proliferative responses of whole PBMC (black columns) and T cell-depleted PBMC (gray columns) are shown for a panel of mycobacterial Ags in three animals, CK556, CP1662, and CP1825. Results are expressed as the mean cpm plus the SEM of triplicate cultures. *, Positive response of -depleted PBMC to Ag compared with the background -depleted PBMC response in medium (99% confidence limits).

View this table: [in this window] [in a new window]   Table 2. Percentages of total TCR+, CD4+, and CD8+ T cells present in the PBMC of each animal

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

In this report we describe the responses of purified bovine T cells to a number of defined recombinant mycobacterial protein Ags. The Ags eliciting the strongest responses (apart from bovine tuberculin) were Ag85 and ESAT-6, both of which have previously been shown to induce strong PBMC and CD4⁺ T cell proliferation responses in M. bovis-infected cattle (27, 39). Other, relatively weak proliferative responses were also consistently present (in 6/7 animals) to the Ags MPB83 and hsp16.1. All of the above Ags plus others (MPB70, MPB64, hsp70) were recognized by T cells from the M. bovis field reactor, probably due to the longer term infection of this animal. The responses of each animal were also likely to contain an element of individual variation that is to be expected in animals of different genetic backgrounds. No Ag-specific T cell responses were detected in control M. avium-sensitized animals. Therefore, T cell responses observed in M. bovis-infected cattle were considered to be M. bovis-specific, and to support a T cell memory in cattle, as described by Hoft et al. (40) for humans vaccinated with BCG.

We measured the levels of cytokines IFN-, IL-2, and TGF- in the supernatants of proliferating T cell, PBMC, and -depleted PBMC cultures. Our choice of cytokines was based upon the numerous publications that support the involvement of these cytokines both in tuberculosis and in the reported activities of T cells. For example, IL-2 is considered to be an important cytokine for T cell activation (41, 42, 43, 44, 45). However, we were able to detect only very low levels of IL-2 that were consistent in all supernatants ( T cell plus feeder, feeder cell-only, or -depleted PBMC) and did not appear to be affected by the presence or absence of Ag.

In contrast to IL-2, the cytokines IFN- and TGF- were both readily detected in culture supernatants. Furthermore, IFN- and TGF- responses were enhanced in some proliferating T cell cultures compared with their respective feeder-only controls. This was most noticeable with those Ags that induced the strongest T cell proliferation, i.e., PPD-M, ESAT-6, and Ag85, suggesting either that bovine T cells were themselves stimulated to produce IFN- and TGF-, or that the proliferation of T cells in the cultures induced an enhanced cytokine production by other cells in the feeder population. The production of IFN- by mycobacteria-responsive human T cells is well documented (44, 45, 46), and T cells have even been described as more efficient producers of IFN- compared to CD4⁺ T cells (45). The main source of TGF- is thought to be activated monocytes and macrophages (47, 48), although it is recognized that other cells such as platelets (49) and T cells (50) may also produce this cytokine. TGF- has been demonstrated to inhibit T, NK, and B cell function, reduce MHC expression, down-regulate proinflammatory cytokines such as IFN-, TNF-, IL-6, IL-1 (51), and IL-2 (50), and to enhance the intracellular growth of M. tuberculosis in human monocytes (52).

Immunomodulation of T cells by T cells was first described in cattle infected with Mycobacterium avium paratuberculosis (53), where the in vitro depletion of T cells was demonstrated to enhance CD4⁺ T cell proliferation to M. a. paratuberculosis Ag. However, M. a. paratuberculosis and M. bovis cause two very different diseases in cattle. M. a. paratuberculosis is the agent of Johne’s disease of the intestine (54), whereas M. bovis affects the upper respiratory airways and associated lymph nodes. It is also becoming increasingly apparent that the cellular immune responses in cattle following experimental infection with these two organisms are different (38, 53). Therefore, it is interesting that cattle T cells, activated by mycobacterial Ag, can down-modulate T cell responses in both infections.

Similarly, a guinea-pig model of M. tuberculosis infection showed that the depletion of FcR⁺ T cells enhanced the proliferative response of splenic lymphocytes to mycobacterial PPD and also to the heat shock proteins hsp65 and hsp70, although unlike bovine T cells, guinea-pig FcR⁺ T cells themselves did not proliferate in response to these Ags (55). Possible mechanisms suggested for T cell modulatory effects thus far have included the cytotoxic activity of T cells against CD4⁺ T cells in the case of bovine paratuberculosis, and the presence of circulating immune complexes, which could modulate FcR⁺ T cells in the guinea-pig.

Our results of T cell modulation in M. bovis-infected cattle have some similarity with these previous reports in that the removal of T cells from PBMC in half the animals tested allowed an increased Ag-specific proliferation to occur. Preliminary results in our laboratory show no corresponding increase in IL-2 in these cultures, suggesting that IL-2 was not responsible for the increase in proliferation of -depleted PBMC observed. Neither did the removal of T cells from PBMC appear to affect Ag-specific IFN- responses. However, preliminary data from our one chronically-infected animal (CK556) suggest that T cells may be able to suppress TGF-, thus supporting a host-protective role for T cells, which could act by causing a delay in the onset and/or severity of disease progression. This is currently under investigation in our laboratory.

In summary, this report describes the Ag-specific proliferation of peripheral blood T cells to defined mycobacterial protein Ags in M. bovis-infected cattle. We further show that T cells are able to suppress Ag-specific T cell proliferation and enhance the production of cytokines IFN- and TGF-. T cells constitute a major T cell population in the peripheral blood of cattle; therefore, the activities of these cells have the potential for a major impact on the outcome of mycobacterial infection.

	Acknowledgments

 
We thank Adam Whelan (Veterinary Laboratories Agency) for providing the recombinant Ags MPB70, MPB83, and ESAT-6. Thanks also go to Dr. M. Singh (GBF, Braunschweig, Germany), Dr. K. Huygen (Institut Pasteur, Brussels, Belgium), Dr. D. Bakker (Animal Health Sciences, Boxtel, The Netherlands), and Dr. J. J. Fourni (Institut National de la Santé et de la Recherche Médicale, France) for supplying the Ags hsp16.1, hsp65, hsp70, 38 kD (M. Singh), Ag85 (K. Huygen), MPB64 (D. Bakker), and the nonprotein ligands TUBag1, Epox-PP, and IHPP (J. J. Fourni). MAbs CC-30, CC-58, and CC-15 were provided by the Institute for Animal Health, Compton, Berkshire, U.K.

	Footnotes

 
¹ This work was funded by the Ministry of Agriculture, Fisheries and Food, U.K.

² Address correspondence and reprint requests to Dr. Shelley G. Rhodes, TB Research Group, Veterinary Laboratories Agency, Addlestone, Surrey KT15 3NB.

³ Abbreviations used in this paper: BCG, bacillus Calmette-Guérin; hsp, heat shock protein.

Received for publication December 1, 2000. Accepted for publication February 20, 2001.

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