'y8 T cells, both human and murine, have been found to be highly responsive to mycobacterial antigens. However, the role and function of 'y8 T cells in the ...
Vol. 60, No. 9
INFECTION AND IMMUNITY, Sept. 1992, p. 3480-3488
0019-9567/92/093480-09$02.00/0 Copyright © 1992, American Society for Microbiology
Role of the Mononuclear Phagocyte as an Antigen-Presenting Cell for Human -y6 T Cells Activated by Live Mycobacterium tuberculosis W. HENRY BOOM,* KEITH A. CHERVENAK, MARIA A. MINCEK, AND JERROLD J. ELLNER Division of Infectious Diseases, School of Medicine, Case Western Reserve University, and University Hospitals, 10900 Euclid Avenue, Cleveland, Ohio 44106-4984 Received 5 March 1992/Accepted 2 June 1992
'y8 T cells, both human and murine, have been found to be highly responsive to mycobacterial antigens. However, the role and function of 'y8 T cells in the immune response to Mycobacterium tuberculosis remain largely unknown. In earlier studies, we demonstrated that monocytes infected with live M. tuberculosis were particularly effective inducers of human peripheral blood 'y8 T cells. The present studies were performed to further characterize the interaction between human mononuclear phagocytes, 'y8 T cells, and live M. tuberculosis, in comparison with CD4+ T cells. First, we found that resting lyb T cells expanded in vitro by live M. tuberculosis were specific for M. tuberculosis, and that heat killing and washing the mycobacteria removed the antigen(s) for yb T cells. In contrast, the heat-killed mycobacteria retained significant antigenicity for CD4+ T cells. Second, live M. tuberculosis-expanded 'yb T cells from healthy tuberculin-positive donors did not respond significantly to the antigens in M. tuberculosis culture filtrate, including the 65- and 71-kDa mycobacterial heat shock proteins. Third, the activation of 'yb T cells by live mycobacteria was dependent on antigen-presenting cells, and mononuclear phagocytes were found to be very efficient antigen-presenting cells both for resting peripheral blood 'yb T cells and for activated expanded 'yB T cells. The mononuclear phagocyte carried the necessary costimulatory factors necessary for -yb T-cell proliferation. Fourth, the antigen repertoire and HLA requirements for CD4+ memory T cells and those for yb T cells appear to be quite distinct from each other. CD4+ T cells recognized both soluble protein antigens and whole organisms in a class II major histocompatibility complex-restricted manner, whereas 'yB T cells appeared to recognize only constituents associated with the whole organism and were not restricted by class I or class II major histocompatibility complex molecules. Finally, the assay system described to expand and purify responding CD4+ and 'yB T cells after stimulation with live M. tuberculosis represented a simple approach to the direct comparison of these two T-cell populations in the interaction with mononuclear phagocytes infected with M. tuberculosis. Such studies provide insight not only into the relative roles of human CD4+ and 'yb T cells in the human immune response to intracellular bacterial pathogens such as M. tuberculosis but also into the basic biologic role of human 'YB T cells in antimicrobial immunity.
Mycobacterium tuberculosis is the etiologic agent of tuberculosis, which remains the leading cause of mortality due to an identifiable infectious agent worldwide (24). The majority of individuals infected with M. tuberculosis successfully contain the primary infection concomitant with the development of a vigorous delayed-type hypersensitivity response (1). The cellular immune response, consisting of T cells and mononuclear phagocytes, is critical to the mediation and regulation of the acquired immune response, which controls primary infection and provides protection against exogenous reinfection (13). Failure of the immune response, as seen in individuals infected with human immunodefi-
response to M. tuberculosis. Recent studies by us and others have demonstrated that ryi T cells may have an important role in the initial human immune response to intracellular pathogens such as M. tuberculosis. The first indication that -yb T cells respond to mycobacterial antigens came from studies of murine -yb T-cell hybridomas, which revealed that a large number of these cells were reactive with mycobacterial antigens (25). Mycobacterial antigens also were found to expand y8 T cells in draining lymph nodes and lungs (3, 19). Furthermore, during primary infection with live Mycobacterium bovis BCG or M. tuberculosis, -yb T cells were found to accumulate at the site of infection (12, 18). In humans, ryi T-cell lines with reactivity to mycobacterial antigens have been derived from the synovial fluid of a rheumatoid arthritis patient, the skin lesions of leprosy patients, and the peripheral blood of a healthy tuberculin reactor (16, 22). In addition, we and others have found that whole intact M. tuberculosis, in contrast to soluble secreted mycobacterial antigens such as the purified protein derivative (PPD) of M. tuberculosis, induce the expansion of human peripheral blood yS T cells (15, 21). In previous studies, we demonstrated that mononuclear phagocytes infected with live M. tuberculosis H37Ra were particularly effective in inducing the expansion of -yi T cells (15). In contrast, mononuclear phagocytes exposed to heat-killed
ciency virus, contributes to progressive primary infection and to reactivation of endogenous foci of mycobacteria. A better understanding of the human immune response to live M. tuberculosis is necessary to develop a sound rationale for the design of a new vaccine or diagnostic reagent. On the basis of studies in humans and animal models, the interaction between CD4+ T cells and mononuclear phagocytes infected with M. tuberculosis traditionally has been considered the principal mechanism responsible for immunologic memory and regulation of the protective immune *
Corresponding author. 3480
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mycobacteria induced a vigorous a,B T-cell (predominantly CD4+) response with little or no -yb T-cell expansion. Our findings and the observations in murine models with live mycobacteria suggest that yb T cells, in addition to CD4+ T cells, may have a key role in the early immune response to mycobacteria. The present studies were performed to further characterize the interaction between human mononuclear phagocytes, -yb T cells, and live M. tuberculosis. We have found that the mononuclear phagocyte is an efficient antigen-presenting cell (APC) for both resting and activated ryb T-cell lines specific for live M. tuberculosis in a major histocompatibility class (MHC)-unrestricted fashion. Furthermore, the repertoire of antigens recognized by CD4+ and -yb T-cell lines expanded by live M. tuberculosis were found to be quite distinct, with ryb T cells responding selectively to the whole mycobacteria and CD4+ T cells recognizing both secreted protein antigens and whole mycobacteria. MATERIALS AND METHODS Bacterial strains, antigens, and cell lines. M. tuberculosis H37Ra was cultured in Middlebrook 7H9, harvested at mid-log phase (A60, 0.5 to 0.8), and frozen at -70°C. Bacterial counts were performed by light microscopy, and viability was determined by counting CFU before and after freezing. Lots were >50% viable after freezing and were used as the source of live M. tuberculosis. Heat-killed (i.e., dead) M. tuberculosis was prepared by autoclaving live M. tuberculosis organisms for 20 min and then washing and resuspending them in RPMI 1640. Live and heat-killed preparations came from the same frozen stock of M. tuberculosis H37Ra (15). Mycobacterium avium MAIS 17 and MAIS 9 were provided by P. Brennan (Ft. Collins, Colo.). PPD of M. tuberculosis was a gift from Lederle Laboratories (Pearl River, N.Y.). A freeze-dried preparation of M. tuberculosis H37Ra (lot no. 783765) was purchased from Difco Laboratories (Detroit, Mich.). The purified recombinant 71-kDa antigen of M. tuberculosis, 64-kDa antigen of M. bovis BCG, native 38-kDa antigen of M. tuberculosis, and recombinant 18-kDa antigen of Mycobacterium leprae were provided by J. van Embden (Bilthoven, The Netherlands) through the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases (2, 6, 17, 30). The purified native 30- to 32-kDa antigen of M. tuberculosis (antigen 6; a-antigen) was provided by T. Daniel (Cleveland, Ohio) (9). Tetanus toxoid was purchased from the Massachusetts State Laboratory Institute (Jamaica Plain). Daudi Burkitt's lymphoma cells were obtained from P. Sondel (Madison, Wis.) (11). Purified fractions A and B from late M. tuberculosis Erdman culture filtrate were provided by I. Orme (Ft. Collins, Colo.). Lipoarabinomannan was obtained from P. Brennan (8). Monocyte isolation and ii T-cell stimulation. Peripheral blood mononuclear cells (PBMC) were obtained by density sedimentation over sodium diatrizoate-Hypaque gradients from individuals known to be either tuberculin skin test positive (PPD+) or to respond vigorously to mycobacterial antigens (PPD) in lymphocyte proliferation assays. PBMC, in RPMI 1640 with 10% pooled human sera, were incubated on plastic tissue culture dishes (Falcon 3003) precoated with pooled human sera for 15 min at 37°C. After 1 h at 37°C, nonadherent cells were removed. After 20 min at 4°C in phosphate-buffered saline, plastic adherent cells (.90% monocytes by Wright's, peroxidase, and nonspecific esterase staining) were collected by scraping with a rubber
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policeman, washed, and counted. Nonadherent cells (2 x 106 per well), as a source of peripheral blood T cells, and monocytes (0.5 x 106 per well) were cultured in 24-well plates (Costar) in 2 ml of complete medium (RPMI 1640, 10% pooled human sera, 20 mM HEPES [N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid], 2 mM L-glutamine, 100 U of penicillin per ml) for 7 to 9 days before the T-cell phenotype was determined. In a modification of an earlier procedure (15), live M. tuberculosis (5 x 106/ml) was added directly to the cultures of T cells and monocytes without a phagocytosis step. Cells were incubated with mycobacteria for 7 to 9 days without the addition of exogenous interleukin-2 to the cultures. These primary yb T-cell stimulations with live mycobacteria will be referred to operationally as resting -yb T-cell induction, where induction is defined as the activation and proliferation of resting -yb T cells present in freshly isolated PBMC. In contrast, expanded activated -yS T cells are the cells present in culture after 7 to 9 days of stimulation with M. tuberculosis. Phenotypic analysis. After 7 to 9 days of coculture, viable cells were harvested and analyzed by indirect immunofluorescence. Cells were stained with a saturating amount of primary monoclonal antibody for 30 min and then with fluorescein isocyanate (FITC)-conjugated goat anti-mouse immunoglobulin G (IgG) (Cappell, Malvern, Pa.). The following monoclonal antibodies were used: OKT3 (anti-CD3, IgG2a) and OKT4 (anti-CD4, IgG2b) (both from Ortho Diagnostics, Inc., Raritan, N.J.); WT-31 (anti-a1 T-cell receptor [anti-ap TCR], IgGl; Becton Dickinson, Mountain View, Calif.; used in initial experiments), replaced with BMA031 (specific for a framework determinant of the a,B TCR, IgG2b; T Cell Sciences, Cambridge, Mass.); TCR-81 (4) (specific for a framework determinant on the 8 chain of the -y8 TCR, IgGl; kindly provided by Michael B. Brenner, Boston, Mass.); and TiyA (specific for V-y9 -yb TCR; IgGl) (31) and bTCS-1 (specific for Vb1 -yS TCR; IgGl) (both from T Cell Sciences) (33). For two-color fluorescence-activated cell sorting (FACS) analysis, phycoerythrin-conjugated OKT-3, FITC-conjugated OKT-4 (Ortho Diagnostics), and FITC-conjugated TCRB-1 (T cell sciences) were used with FITC- and phycoerythrin-conjugated isotypic controls. Cells were analyzed by FACS on a Cytofluorograph IIs (Becton Dickinson) by using the 488-nm argon line at 250 mW and a HEPES-citrate saline sheath (pH 7.4). A filter combination providing 530/22-nm and 610/20-nm band passes (Becton Dickinson) was employed for measuring fluorescein and phycoerythrin, respectively. The cytometer was calibrated for both light scatter and immunofluorescence with Immunobrite beads (Coulter Immunology, Hialeah, Fla.). Cells were gated on a two-parameter plot of 900 versus forward-angle scatter. The gate for lymphocytes was widely set to include small as well as large lymphocytes and to avoid bias towards a particular subpopulation. The gate was kept constant for each experiment. Five thousand gated events were recorded for each cell surface marker. Fluorescence signals were amplified over a 3-decade range. Markers were set visually and kept constant throughout the analysis of each experimental group. The position of the cutoff marker was determined by the distribution of cells stained with FITC-conjugated goat anti-mouse antibody alone. Populations to the left of the marker were considered negative and those to the right were considered positive for a cell surface marker. The percentage reported for a given cell surface marker represents the proportion of gated cells with a fluorescence signal greater than those stained with second antibody alone.
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Purification of 'y& and CD4+ T cells after stimulation with M. tuberculosis. T cells were harvested after 7 to 9 days of stimulation with live M. tuberculosis, and viable cells were purified by density sedimentation over sodium diatrizoateHypaque gradients. Expansion of y1 T cells was confirmed by FACS analysis (see above), and T-cell subsets were enriched by negative selection with magnetic beads coated with antibodies (Dynal, Great Neck, N.Y.). For yb T-cell enrichment, cells were treated simultaneously with antiCD4- and anti-CD8-coated beads. For CD4+ T-cell enrichment, cells were treated first with TCR-81 and then with goat anti-mouse IgG-coated beads and anti-CD8-coated beads. Antibody-coated beads were used at a 10:1 bead-to-cell ratio based on the estimated number of T cells from each T-cell subset (CD4+, CD8+, and -y8 T cells) present after 7 to 9 days of stimulation with live M. tuberculosis. Generally, one cycle of treatment was sufficient for depletion of the T cells, although in some experiments two cycles were performed. The purity of the negatively selected T-cell populations was always assessed by either single- or two-color FACS analysis (see above) before the cells were used in experiments. Purification of small resting T cells. PBMC were isolated as described above, monocytes were removed by adherence, and the nonadherent cells were passed over a nylon-wool column (Fenwall Laboratories, Grove Park, Ill.) which had been preincubated at 37°C in 10% fetal calf serum for 30 min. After a 30-min incubation period at 37°C, lymphocytes were eluted from the nylon-wool column and placed on a discontinuous gradient composed of 40, 43.5, 47, and 54% Percoll (Pharmacia, Uppsala, Sweden). Cells at the 47 to 54% interface were collected, washed, and depleted of any residual class II MHC antigen-expressing cells by treatment with OK-Ial (Ortho Diagnostics) and rabbit complement (PelFreez, Brown Deer, Wis.). The remaining high-density lymphocytes were >90% TCR+ (c43 TCR + 9y TCR) by FACS analysis and completely dependent on the addition of APCs for proliferative responses to recall antigens (PPD, tetanus toxoid) and mitogens (phytohemagglutinin, anti-CD3) (28). Proliferation assay. Purified T cells (2.5 x 104 per well) were cocultured with 5 x 104 nonirradiated monocytes as APCs per well and with antigen for 72 h in 96-well plates. Irradiated Daudi cells as APCs were used at 5 x 104 cells per well. Cells were pulsed with 1 ptCi of [3H]thymidine (ICN, Costa Mesa, Calif.) for 12 to 16 h before being harvested on glass fiber filters with a PHD harvester (Cambridge Technology, Watertown, Mass.). [3H]thymidine incorporation was measured by liquid scintillation counting and expressed as the mean counts per minute. Contamination of the nonirradiated monocyte populations with T cells was monitored in each experiment by having as the controls monocytes alone with or without either PPD, live mycobacteria, or heat-killed mycobacteria. Only those experiments in which these controls had stimulation indices of less than 2 were included for analysis. Cell proliferation was considered significant when the ratio of counts per minute obtained in the presence of antigen was greater than three times the counts per minute present in the absence of antigen.
RESULTS Role of the mononuclear phagocyte as APC. In earlier experiments, mononuclear phagocytes which had phagocytosed live mycobacteria before their addition to peripheral blood T cells were used to induce resting yb T-cell expansion. This primary yb T-cell expansion refers to the activation and proliferation of freshly isolated resting peripheral
PBMC
NAC
NWC
FIG. 1. Progressive depletion of APCs from PBMC for yyb T-cell expansion by live M. tuberculosis and reconstitution with monocytes. T cells (2 x 106 per well) as either PBMC, nonadherent cells (NAC), nylon-wool passed cells (NWC), cells recovered from 54% Percoll (PERCOLL), or anti-DR treated cells (DR-DEPL.) were stimulated with mycobacteria for 7 days with and without autologous monocytes (5 x 105 per well). The percentages of -y T cells were determined by FACS analysis and are expressed as the percentages of viable cells.
blood 9y T cells, in contrast to the activated 91 T cells (short-term yyb T-cell lines) present in in vitro cultures after 7 to 9 days of stimulation with M. tuberculosis. To demonstrate that the primary 9yb T-cell induction was indeed dependent on APCs and that mononuclear phagocytes could serve efficiently in this capacity, high-density peripheral blood T cells were purified and rendered APC dependent for protein antigen and mitogen responses as described in Materials and Methods. For these experiments, a modification of the initial protocol was necessary. The separate phagocytosis step was eliminated, and live mycobacteria (5 x 106/ml) were added directly to the cell cultures. As shown in Fig. 1, as the purification of resting T cells proceeded, the ability of M. tuberculosis to induce 9y1 T cells was progressively reduced from 27.6 to 1.7%, demonstrating that live mycobacteria were not capable of directly stimulating resting 91 T cells in the absence of APCs. The addition of autologous monocytes (5 x 105 per 2 x 106 high-density T cells) reconstituted the primary -yb T-cell expansion. Once this APC-dependent system for primary 9y T-cell expansion by live M. tuberculosis had been established, the efficiency and HLA restriction requirements of the monocyte for 9y1 T-cell expansion were studied. As shown in two representative experiments in Fig. 2, 1 x 104 to 5 x 104 autologous monocytes reconstituted the 9y T-cell expansion for 2 x 106 purified T cells (2 to 10% 9y TCR+). In some experiments, fewer than 104 monocytes reconstituted the 98 T-cell response (Fig. 2B). The maximal response was induced by 1 x 105 to 5 x 105 monocytes. When monocytes and purified T cells from HLA-A, -B, -C, and -DR mismatched donors were used, efficient 9y1 T-cell induction was seen (Table 1). To assure that the -yi T-cell induction was not due to contaminating T cells present in the monocyte preparation, irradiated monocytes were used (Table 1, experiment 1). These results indicate that monocytes are effective and efficient APCs for the activation and proliferation of resting 91 T cells by live M. tuberculosis. In addition, our studies with resting 9y1 T cells confirm the findings of others with activated 98 T-cell populations of various specificities concerning the lack of restriction by class I and II MHC molecules on APCs.
MACROPHAGES, -yb T CELLS, AND M. TUBERCULOSIS
VOL. 60, 1992 rA
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FIG. 2. Resting T cells (2 x 106 per well) were cultured with various numbers of autologous monocytes (0 to 106 per well) and live M. tuberculosis for 7 days. The percentages of -yl T cells were determined by FACS analysis and are expressed as the percentages of viable cells after 7 days of stimulation with mycobacteria.
Antigen specificity of live M. tuberculosis-activated -y8 T cells. To further characterize the activated yb T cells expanded by live M. tuberculosis, -yb T cells were enriched by removing CD4+ and CD8+ T cells from the lymphocyte cultures exposed to M. tuberculosis for 7 to 9 days. For enrichment by negative selection, experiments in which the y8 T cells expanded by live M. tuberculosis constituted at least 25% of the viable cells were performed. Generally, one cycle of negative selection was sufficient, resulting in a lymphocyte population which was 275% CD3+ (range, 75 to 90%), the same percentage of yb TCR+, and less than 5% CD4+ and CD8+ T cells as measured by single-color immunofluorescence. The remaining 15 to 25% CD3- cells consisted of CD19+ B cells, CD16+ NK cells, and M1+ mononuclear phagocytes. To confirm the purity of these negatively selected T-cell populations, two-color FACS analysis was performed, using FITC-conjugated TCRb1 and OKT4 and phycoerythrin-conjugated OKT3. A representative experiment of five performed is shown in Fig. 3, in which 99% of the CD3+ T cells were -yb TCR+ and 1% were CD4+ (Fig. 3A and B). The reciprocal selection on the same starting population is shown in Fig. 3C and D. CD8+ and yB T cells were removed, resulting in a CD3+ population which was 99% CD4+. Since the majority (.60%) of peripheral blood -yb T cells carry a TCR utilizing V-y9 (as recognized by the Ti-yA antibody) usually in association with V82 and the second most common subset (10 to 20%) utilizes Vb1l (recognized by bTCS1) for its TCR, we were interested in determining which subset was expanded by live M. tuberculosis. The yb
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T cells in our experiments were >95% V-y9' and
