Flowcytometric assessment of intracellular interferon -γ production in human CD4 + ve T-cells on mycobacterial antigen stimulation

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1 J. Biosci., Vol. 22, Number 1, January 1997, pp Printed in India. Flowcytometric assessment of intracellular interferon -γ production in human CD4 + ve T-cells on mycobacterial antigen stimulation S BHATTACHARYYA, S Ν DAS, Α Β DEY*, Κ NAGARKAR*, J LOBO**, S Κ KAPOOR** and Η Κ PRASAD Department of Biotechnology, *Department of Medicine,**Center for Community Medicine, All India Institute of Medical Sciences, New Delhi , India MS received 6 February 1996; revised 22 July 1996 Abstract. Interferon- (IFN-γ ) has been considered to be a critical protective immunomodulatory component against Mycobacterium tuberculosis (M. tb.) infection. In this study T-cell proliferation and IFN-γ production upon stimulation with M. tb. were assessed in patients of pulmonary tuberculosis and healthy contacts. The studies were based on lymphocyte transformation test and detection of intracellular IFN-γ production by CD4 + ve T-cells by flowcytometry. Patients showed lower levels of proliferation, the stimulation index being in the range of (mean + SD) compared to the contacts (SI = 4 59± 1 6) (P < 0 01). The kinetics of intracellular induction of IFN-γ on M. tb. stimulation showed a proportional increase in the CD4 + ve T-cell population. The increase was maximal between h of culture. In healthy contacts the number of IFN-γ expressing CD + ve T-cells increased to 2 5 to cells/ml in M. tb. stimulated cultures compared to control cultures ( ). In contrast patients showed no/marginal increase in CD4 + ve T-cell population expressing intracellular IFN- γ Thus the lack of induction of IFN in CD4 + ve T-cells in patients could be a critical shortcoming in their ability to combat tubercle bacilli infection. Keywords. Interferon-γ; intracellular; mycobacteria; tubercullosis; flowcytometry. 1. Introduction Generation and secretion of cytokines is the hall mark of activated immunocompetent cells. Several techniques are currently in vogue to monitor cytokines at the level of: (i) mrna by RT-PCR (Johnson and McMurray 1994), (ii) secreted protein in culture supernates by ELISA (Barnes et al 1993) or bioassay (Curry et al 1987) and at the level (iii) of a single cell. Detection of cytokines at the single cell level has been exploited by several workers. The contribution of a defined population of cells and the expression of cytokines has been examined, using monoclonal antibodies for the detection of intracellular expression of cytokines and cell surface markers (Sander et al 1991; Jung et al 1993; Assenmacher et al 1994). Corresponding author (Fax, , ; , hkp@aiims.ernet.in). Abbreviations used: M. tb., Mycobacterium tuberculosis (H37Rv); IFN γ interferon- γ; LTT, lymphocyte transformation test; S.I., stimulation index; PBS, phosphate buffer saline; DPBS, Dulbecco's phosphate buffer saline; PFA, paraformaldehyde; FACS, fluorescence activated cell sorter; PHA, phytohaemaglutinin; PBMC, peripheral blood mononuclear cell; BD, Becton Dickinson; FCS, fetal calf serum; SA-PE, streptavidin-phycoerythrin; FITC, fluorescein isothiocyanate. 99

2 100 S Bhattacharyya et al The present study was undertaken to assess the frequency of interferon- γ (IFN-γ) producing CD4 + ve T-cells in human peripheral blood mononuclear cell (PBMC). The cells isolated from healthy contacts and patients of pulmonary tuberculosis were co-cultured in vitro with or without heat killed tubercle bacilli. The intracellular expression of IFN-γ in CD4 + ve T-cells was assessed by fluorescent microscopy and flowcytometry. 2. Materials and methods 2.1 Subjects Eight patients attending the out-patient clinic at the All India Institute of Medical Sciences, diagnosed for pulmonary tuberculosis, and an equal number of healthy contacts were included in the study. The healthy contacts were screened for clinical signs and symptoms of tuberculosis, followed by chest X-ray and staining of acid fast bacilli in sputum. 2.2 Reagents Mycobacterium tuberculosis H37Rv heat inactivated bacilli was kindly provided by Dr Patrick Brennan, Colorado State University, USA. Ficoll-paque from Pharmacia (Uppsala, Sweden) was used for PBMC separation. Cells were cultured in RPMI-1640 with 25 mm HEPES (GIBCO, Grand Island, NY, USA). Anti-CD4 antibody was affinity purified from mouse hybridoma CRL 8002, ECACC, and biotinylated (Harlow and Land 1988). Streptavidin-phycoerythrin (SA-PE) was purchased from Becton-Dickinson, USA. Anti-human-interferon-γ supernatant derived from murine hybridoma (ATCC No. HB-8291, IgG 1) was kindly provided by Dr S Rath, National Institute of Immunology, New Delhi. Anti-mouse-FITC was purchased from DAKO, Denmark. [ 3 H] thymidine (low specific activity) was obtained from the Bhabha Atomic Research Centre, Bombay. Phytohaemagglutinin (PHA) and saponin were obtained from GIBCO and Sigma respectively. 2.3 Stimulation of PBMC Ficolled PBMC (10 6 /ml, Boyum 1968) were suspended in RPMI-1640, enriched with 10% autologous serum. One hundred µl of cell suspension was distributed into 96 well round bottom tissue-culture plates (Flow Laboratories, Scotland, UK). The PBMC were incubated with 25 µl suspension of M. tb. bacilli ( bacilli/ml) or PHA. The cultures were incubated at 37 C, 5% CO 2 for 5 days. 2.4 Lymphocyte transformation test The microtitre plate cultures cells were pulsed on the 5th day with 0 5 µci of [ 3 H] thymidine for 18 h before being harvested on glass fiber filters using a cell harvester (PHD, Cambridge Technology, Watertown, Mass, USA). [ 3 H] thymidine incorporation was measured by liquid scintillation counting (Rack Beta counter, LKB,

3 Flowcytometric detection of intracellular interferon γ 101 Pharmacia United, Sweden). The incorporation of [ 3 H] thymidine was expressed as stimulation index, and was calculated as follows: 2.5 Assessment for intracellular IFN production by fluorescent microscopy and flowcytometry Cultured PBMC with and without antigen were washed with PBS ph 7 4 at 8 C, 200 g for 10 min. The cells were suspended in 50 µl of staining buffer (0 1% FCS in DPBS). It was stained for CD4 surface marker by successive incubation with biotinylated-anti CD4 followed by SA-PE. The stained cells were fixed in 4% paraformaldehyde for 20 min on ice, followed by washing and suspension of cells in permeabilization buffer (0 1 % saponin in staining buffer). The detection of intracellular IFN-γ was done, by successive incubation with anti-human IFN-γ (1:10) and antimouse FITC (1:50). The dual stained cells were stored in 0.1% PFA at a concentration of 10 6 cells/ml for further analysis by fluorescent microscopy (Zeiss, Germany) and flowcytometry. Ten-20 random fields were examined. Each field was viewed in sequence first under visible light, followed by UV light optimized for FITC fluorescence and finally by filters optimized for PE. For each sample the number of dual labelled fluorescent cells was enumerated per 10 6 PBMC. The following filters were used for FITC, excitation filter BP , and the barrier filter LP 520; and excitation filter BP 546 and barrier filter LP 590 for PE. For flowcytometry the FACScan flowcytometer (BD) and Lysis II software (BD) were used for cell acquisition and analysis of the results. The lymphocyte gated population was analysed using a second gate set for CD4 + ve cells (PE). The frequency of the CD4 + ve T-cells expressing IFN-γ were determined in the gated cell population. 3. Results 3.1 Lymphoproliferative response of patients and healthy contacts to M. tb. antigen An aliquot of PBMC isolated from patients and contacts were cultured with M. tb. antigen for assessing the classical lymphoproliferative responses. The results expressed as stimulation index are shown in table 1. The patients exhibited limited proliferative responses (2 17±1 1) compared to healthy contacts (4 59±1 6), (P < 0 01). The results were subsequently compared with the frequency of CD4 + ve T-cells expressing intracellular IFN-y, 3.2 Induction of IFN-γ in PH A activated CD4 + ve T-cells Ficolled human PBMC activated with or without PHA were stained for mitogen induced intracellular expression of IFN-γ. The cultures were terminated and stained at

4 102 S Bhattacharyya et al Table 1. Lymphoproliferative response a to M.tuberculosis. a PBMC from patients and healthy contacts were cultured with M. tb. for 5 days. On the 5th day the cultures were pulsed for 18 h with [ 3 H ] thymidine andharvested. b Mean ± SD. C [ 3 H] thymidine incorporation expressed as mean stimulation index ± SD. d PBMC cultured devoid of M. tb. e PBMC cultured with M. tb. f Number of samples. S.I. of patients vs healthy contacts Ρ < h for CD4 surface marker followed by intracellular staining for IFN-γ The dual stained CD4+veandI FN-y expressing cells were detectable by microscopy (table 2) in all the healthy contacts examined. The number of dual stained cells ranged between PBMC/ml. Table 2. Expression of IFN -y in PHA stimulated CD+re T-cells. *Not Detectable C, Control cultures devoid of PHA; S, stimulated cultures incubated with PHA. PBMC were cultured with or without PHA for 72 h. Dual staining for intracellular IFN and CD4 membrane marker was carried out. The stained cells were analysed using a fluorescent microscope.

5 Flowcytometric detection ofintracellularinterferon-γ Specificity of the detection of intracellular expression of IF-γ in PH A stimulatedcells In order to evaluate the specificity of staining technique used for the detection of intracellular IFN in CD4 + ve T-cells the following controls were used: (i) staining of non-permeabilized (figure 1A) and permeabilized (figure 1B) cells with anti-ifn-γ murine monoclonal antibody, (ii) staining of permeabilized cells with the secondary antibody i.e., rabbit anti-mouse FITC conjugate (figure 1C) and (iii) isotype control i.e., non-specific murine antibody conjugated to FITC and PE (figure 1D). The dot plots in figure 1(A, B, C) represents the data on IFN expressing cells in the CD4 gated population analysed using the gates described in 2 5. Comparing the Figure 1. Gated dot-plot of lymphocytes showing specificity of intracellular IFN-γ staining in CD4+ve T-cell subset. PBMC (10 6 /ml) isolated from healthy contacts were cultured with a suspension of M. tb. (heat inactivated, bacilli/ml) for 5 days. The cells were double stained for CD4 surface marker and intracytoplasmic IFN-γ (A) Shows the results of staining with non-permeabilized cells. (B) An aliquot of cells permeabilized and stained. (C and D) Results obtained on staining of permeabilized cells with rabbit anti-mouse FITC conjugate and isotype control with irrelevant murine antibody conjugated to FITC and PE. The dot-plots in (Α, Β and C) have been obtained from the lymphocyte gated population analysed using a second gate for CD4 + ve T-cells (PE). The dot-plot in (D) was obtained from the lymphocyte gated population alone.

6 104 S Bhattacharyya et al frequency of cells detected in figure 1A with B, demonstrates that only with permeabilized cells (54 2%) the flowcytometric signal for intracellular cytoplasmic IFN-γ was detected. However a limited number of cells (7%) were detected with membrane bound IFN-γ (figure 1A). The results shown in C compared with Β demonstrate the specificity of the staining observed exclusively with IFN-γ murine monoclonal antibody. The dot lot in figure 1D was obtained using the lymphocyte gated population only. The absence of PE/FITC labelled cells using non-specific murine antibodies conjugated to FITC and PE in the panel demonstrates the specificity of the labelling of human PBMC with murine monoclonal antibodies speicific for CD4 and IFN-γ. 3.4 Expression of IFN-y in M. tb.stimulated CD4+ ve human T-cells 3.4a Kinetics of the induction of IFN-γ in M. tb. stimulated CD4 + ve T-cells: Human PBMC (10 6 /ml) were co-cultured with heat inactivated M. tb. ( /nil) over a 5 day period. The cultures were terminated at 24 h time intervals. The harvested cells were Figure 2. Kinetics of IFN-γ expression in CD4 + ve T-cells derived from 3 healthy contacts (,, ). Aliquots ofcultured cells at 24, 48, 72, 96 and 120 h after incubation were harvested, fixed, permeabilized and stained for cytoplasmic IFN and for CD4 on the cell surface.

7 Flowcytometric detection ofintracellularinterferon-γ 105 processed for the detection of cytoplasmic IFN-y expression in CD4 + ve T-cells. The frequency of the CD4 + ve T-cells expressing IFN in culture has been depicted in figure 2. The number of cells detected proportionally increased with the duration of the culture. Maximum number of dual stained cells were detected by 96 and up to 120 h, in the 3 individuals examined. The maximum number of cells detected ranged between 1 7 to CD4 + ve T-cells. Subsequently the antigen stimulated were terminated between h of culture. 3.4b Healthy contacts: PBMC isolated from healthy individuals were co-cultured with M. tb. antigen as described earlier. The stimulated cultures were stained and analysed for the intracellular expression of IFN-γ in CD4 + ve T-cells. The results are shown in figure 3. The representative data of a healthy contact (figure 3A,C) clearly shows the presence of dual stained CD4+ve T-cells in PBMC stimulated with mycobacterial antigen (figure 3C) Figure 3. Cytoplasmic expression of IFN-γ in CD4 + ve T-cells derived from healthy contacts (A and C) and patient (B and D). The isolated PBMC were co-cultured with M. tb. antigen (C and D). (A and B) represent control cultures of healthy contact and patient respectively. The cells were harvested on the 5th day stained for CD4 cell surface marker, fixed, permeabilized and stained for intracellular IFN The dot-plots were obtained after analysis of the lymphocyte gated population with a second gate for CD4 + ve T-cells (PE).

8 106 S Bhattacharyya et al compared to control cultures (figure 3 A). All the individuals exhibited enhanced expression of IFN-γ in CD4 + ve T-cells. The number of CD4 + ve T-cells detected ranged from in M. tb. stimulated cultures, compared to in controls (figure 4). Figure 4. Frequency of IFN expressing CD4+ve T-cells in control ( )and M. tb. stimulated ( bacilli/ml) ( )in human PBMC derived from healthy contacts. 3.4c Patients of pulmonary tuberculosis: PBMC isolated from patients were cultured in identical conditions and processed on the 5th day for intracellular expression of IFN-γ in the CD4 + ve T-cell subset. The dot plot of a patient has been shown in figure 3B, D. This representative data shows the absence of intracytoplasmic expression of IFN-γ in CD4+ve T-cells in the presence of heat-killed tubercle bacilli (figure 3D) compared to control cultures (figure 3B), In contrast to the enhanced frequency of CD4+ve T-cells expressing IFN in contacts, no/marginal increase in this subset of T-cells expressing IFN -γ was seen in patients ( ) (figure 5). 4. Discussion This is a preliminary report of the detection of IFN producing cells in the CD4 + ve T-cell subset by flowcytometric analysis. This technique permits the simultaneous

9 Flowcytometric detection of intracellular interferon-γ 107 Figures 5. Frequency of IFN-γ expressing CD4 + ve T-cells in control ( ) and M. tb. stimulated PBMC ( ) derived from patients of tuberculosis. detection of I FN-γ along with a cell surface marker (CD4). In all, 8 patients and an equal number of healthy contacts were analysed for frequency of CD4 + ve T-cells expressing IFN-γ. Initial standardization experiments were conducted to monitor the intracellular expression of IFN-γ in PHA stimulated PBMC. The enhanced expression was only detected in mitogen stimulated cultures. The intracellular fluorescence seen in these experiments was specific and related to cytoplasmic expression of IFN-γ as: (i) non-stimulated cells had lower or no intracytoplasmic fluorescent cells, (ii) the intracellular fluorescence was detectable only in permeabilized cells and was not due to internalization of the monoclonal antibody and (iii) the intracytoplasmic IFN was detected with the anti-ifn-γ monoclonal antibody exclusively. Healthy contacts in general had elevated numbers of CD4+ve T-cells expressing IFN-γ compared to PBMC derived from patients. This feature was seen both in stimulated as well as in cultures devoid of antigen. The results of the study showed that healthy individuals in vitro showed enhanced frequency of CD4 + ve T-cells expressing IFN-γ in response to M. tb. antigen ( ) compared to control cultures ( ). As the study was limited to examining circulating CD4 + ve T-cells derived from peripheral blood, this feature needs to be confirmed by examining cells isolated from lesions, before a conclusion could be drawn on the ability/inability of patients to generate IFN-γ expressing cells.

10 108 S Bhattacharyya et al Besides CD4 + ve T-cells other cells such as CD8 + ve and T-cells and NK cells are known to produce IFN-γ on encountering mycobacterial antigens (Hahn and Kaufmann 1981; Munk et al 1990; Bancroft et al 1991; Orme et al 1992). The ability of these subpopulation of cells to generate IFN-γ in patients is being investigated. Immunity to mycobacteria is mediated by the activation of sensitized T-cells associated with the secretion of a host of cytokines. Functional role of T-cells and its subsets is being actively examined. Both CD4 and CD8 have been shown to play a protective role in the murine system for combating M. tuberculosis challenge (Orme 1987). In contrast mice infected with atypical mycobacteria the protective role of CD8 + ve T-cells has been shown to the minimal/absent (Saunders and Cheers 1995). Indicating that the T-cell response generated following infection with M. tuberculosis is different from the response generated by atypical mycobacterial infection. Among the cytokines generated by sensitized T-cells the role of IFN-γ has been investigated extensively. IFN-γ modulate several immune mediated functions (Flynn et al 1993; Sanchez et al 1994; Surcell et al 1994). It has been considered critical in generation of toxic oxygen and nitrate radicals associated with the killing and clearance of intracellular mycobacteria from infected macrophages (Chan et al 1992; Rook et al 1986). In vivo knockout mice that lack IFN-γ/theIFN-γ receptor genes confirmed the critical role of IFN-γ in the generation of protective cellular immunity to M. tb. infection (Cooper et al 1993; Kamijo et al 1993). Though the contribution of IFN-γ is important, the influence of other cytokines and their source needs to investigated in patients and contacts to make a meaningful conclusion on the critical role played by cytokines in combating mycobacterial infection in humans. Acknowledgements This work was supported by the Department of Biotechnology and Council of Scientific and Industrial Research, New Delhi. SB is a recipient of the U G C fellowship. Technical help was provided by Mrs A John, Mr Ρ Μ Manickam and Dhanpal Singh. We thank Ms Vanilla Bhasin and Mr Bhavneet Singh of BTIS for their help and co-operation. References Assenmacher Μ, Schmitz J and Radruch A 1994 Flowcytometric determination of cytokines in activated murine Τ helper lymphocytes: expression of interleukin-10 in interferon- and interleukin-4 expressing cells; Eur. J. Immunol Bancroft G J, Schreiber R D and Unanue Ε R 1991 Natural immunity: a T-cell-independent pathway of macrophage activation defined in scid mouse; Immunol. Rev Barnes Ρ F, Lu S, Abrams J S, Wang E, Yamamura Μ and Modlin R L1993 Cytokine production at the site of disease in human tuberculosis; Infect. Immun Boyum A 1968 Isolation of mononuclear cells and granulocytes from human blood; Scand. J. Clin. Lab. Invest. (Suppl. 97) 21 77'-89 Chan J, Xing Y, Magliozzo R S and Bloom Β R 1992 Killing of virulent Mycobacterium tuberculosis by reactive nitrogen intermediates produced by activated murine macrophages; J. Exp. Med Cooper A M, Dalton D K, Stewart Τ A, Giffin J Ρ and Orme I M 1993 Disseminated tuberculosis in interferon-τ gene-disrupted mice; J. Exp. Med

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