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. 2004 Jan;111(1):100–106. doi: 10.1111/j.1365-2567.2003.01785.x

Intracellular expression of interleukin-4 and interferon-γ by a Mycobacterium tuberculosis antigen-stimulated CD4+ CD57+ T-cell subpopulation with memory phenotype in tuberculosis patients

Maria C Jiménez-Martínez *, Marisela Linares *, Renata Báez *, Luis F Montaño †, Salvador Martínez-Cairo ‡, Patricia Gorocica *, Raúl Chávez §, Edgar Zenteno §, Ricardo Lascurain §
PMCID: PMC1782398  PMID: 14678204

Abstract

In some chronic pathological conditions, antigen persistence activates and expands the CD4+ CD57+ T-cell subset. The host immune response against tuberculosis infection is maintained through the continuous presence of antigen-stimulated effector/memory helper T cells. To determine whether CD4+ CD57+ T cells were also expanded in human tuberculosis, we analysed (by flow cytometry) the phenotype of peripheral blood CD4+ T cells from 30 tuberculosis patients and 30 healthy controls. We observed a significant increase in the CD4+ CD57+ T-cell subset in tuberculosis patients in comparison to healthy controls (P < 0.001). Most CD4+ CD57+ T cells exhibited a CD28− CD45RO+ CD62L− phenotype, which is associated with memory cells. In vitro, a higher number of antigen-stimulated CD4+ CD57+ T cells produced intracellular interferon-γ and interleukin-4 compared with antigen-stimulated CD4+ CD57− T cells (P < 0.001). These findings suggest that the majority of CD4+ CD57+ T cells correspond to a phenotype of activated memory T cells.

Introduction

The resurgence of tuberculosis (TB) has led to a re-examination of the host T-cell immune response in the control of Mycobacterium tuberculosis infection.1–3 In animal experimental models, recognition of infected macrophages by a subset of memory T cells is an event of crucial importance in acquired resistance towards a secondary TB infection.4,5 In these models, the protective immune response to TB is carried out by effector/memory CD4+ T cells with the CD44hi CD45RBlo D62L− phenotype, which produce significant quantities of cytokines.4–6 Shift to the memory T-cell phenotype depends on the persistent and repetitive exposure to mycobacterial antigen.7,8 There are some pathological conditions in which antigen persistence is associated with the expansion of a CD4+ T-cell subset bearing the CD57 marker, i.e. human immunodeficiency virus (HIV) infection,9 renal or bone marrow allograft transplant,10,11 chronic lymphocytic leukaemia,12,13 colorectal cancer,14,15 rheumatoid arthritis16,17 and malarial infection.18 CD57 is the sulfated polysaccharide SO4-3GlcAβ1,3Galβ1,4-GlcNAc (also known as HNK-1 or Leu-7), which is present on several cell-surface glycoproteins and glycolipids19,20 and on unconventional T cells.10,18 It has been reported that CD57-bearing glycolipids are ligands for L-selectin and P-selectin,21 interleukin (IL)-622 and nervous system proteoglycans.23 The expression of CD57 on T cells has been suggested as a marker of late memory T cells,24,25 but the true functional significance of this cell subpopulation is uncertain. The aim of this work was to determine the frequency and characteristics, in a typical antigen-persistent pathological condition (such as active pulmonary TB) of the CD4+ CD57+ T cells and their cytokine profile.

Materials and methods

Patients

Thirty adult individuals (all native and residents of Mexico City), with active pulmonary TB and reactors to intradermal tuberculin purified protein derivative (induration >10 mm after 72 hr), were studied. Pulmonary TB diagnosis was based on clinical history, physical examination, chest X-rays, and positive detection of acid-fast bacilli in sputum as well as isolation and typification of mycobacteria in sputum cultures. According to the diagnostics standards of the American Thoracic Society, all patients were classified as having TB class 3 category I disease.26 Blood and stool cultures were performed for all patients in order to eliminate possible bacterial or parasitic co-infections. After obtaining informed consent, and before treatment, a peripheral blood sample was obtained from each individual. Thirty clinically age-matched healthy volunteers were used as controls. All patients gave informed consent for blood sampling and tuberculin skin testing after written information was provided. The Medical Ethics Committee of the National Institute of Respiratory Diseases, Mexico City, approved the study protocol.

Monoclonal antibodies and reagents

Phycoerythrin (PE)-labelled mouse immunoglobulin G (IgG) monoclonal antibodies (mAbs) to human CD28, T-cell receptor (TCR)-γδ and CD44, as well as fluorescein isothiocyanate (FITC)-labelled antibodies to human CD62L, CD69 and TCR-αβ, and CyChrome-labelled streptavidin, were from PharMingen (San Diego, CA). Mouse immunoglobulin M (IgM) mAbs to human CD57, and Cy3-labelled goat anti-mouse IgM antibody, were from Zymed Laboratory (San Francisco, CA). FITC-labelled goat anti-mouse IgM, FITC-labelled mouse anti-human CD4 and CD45RA, and PE-labelled mouse anti-human CD4 and CD45RO, were from Southern Biotech Inc. (Birmingham, AL). PE-labelled antibodies to human IL-4 were from Becton Dickinson (San Jose, CA). Biotin-labelled rat anti-mouse IgM, and FITC-labelled antibodies to human CD14, CD19 and IFN-γ, were from Serotec Inc. (Raleigh, NC). The CD4+ T-cell-negative isolation kit and magnetic microbeads coated with antibodies to mouse IgM for use in the magnetic antibody cell sorting (MACS) system were from Miltenyi-Biotech (Bergisch Gladbach, Germany). Lymphoprep (Ficoll 1.077 density) was from Nycomed Pharma As. (Nyegaard, Oslo, Norway). Concanavalin A (Con A), saponin, brefeldin-A, RPMI-1640, and salts were purchased from Sigma Chemical Co. (St Louis, MO). Sodium pyruvate, l-glutamine and 2-mercaptoethanol were from Gibco BRL. (Rockville, MD, USA). Fetal calf serum (FCS) was from HyClone Laboratories (Logan, UT). Soluble culture filtrate protein extracts were obtained from M. tuberculosis H37Rv strain (ATCC 27294) according to Parra et al.27

Peripheral blood mononuclear cells

Whole heparinized peripheral blood was diluted 1: 2 (vol/vol) in phosphate-buffered saline (PBS), pH 7.2. Peripheral blood mononuclear cells (PBMC) were separated on a Ficoll density gradient by centrifugation (30 min, 500 g, 16°).28 After centrifugation, the interface cells were collected, washed twice and counted in a haemocytometer, assessing its viability by Trypan Blue dye exclusion. Then, cells were resuspended in PBS at a concentration of 107 cells/ml.

Purification of monocytes and T-cell subsets

Monocytes were isolated from PBMC by adhesion for 2 hr in six-well flat-bottomed tissue culture plates (Costar, Cambridge, MA) in RPMI-1640 at 37° in a 5% CO2 humidified atmosphere. After incubation, non-adhered cells were removed and adhered cells were recovered from the culture plate by washing with cold PBS containing 0.05 mm EDTA.29 Then, the cells were washed, suspended in culture medium and counted in a haemocytometer, determining their viability by Trypan Blue dye exclusion. The phenotype of the adherent cells was analysed by flow cytometry using FITC-labelled anti-CD14 and anti-CD19 mAbs; the proportion of CD14+ adherent cells was always >85%.

CD4+ T cells were isolated from the non-adherent cell supernatants, or from PBMC, by negative magnetic selection using microbeads coated with antibodies to human CD8+, CD11b+, CD16+, CD19+, CD36+ and CD56+ in a magnetic activated cell sorting system, according the manufacturer's instructions; the magnetically labelled CD4− T cells were retained in the column, while unlabelled CD4+ T cells ran through.

Cell co-cultures

CD4+ T cells were co-cultured with their autologous monocytes (CD14+ adherent cells), at a 5: 1 ratio (2 × 105 cells/well), in 96-well flat-bottomed cell culture plates (Costar) in RPMI-1640 supplemented with 1 mm sodium pyruvate, 2 mm l-glutamine, 50 µg/ml gentamicin and 0.5% heat-inactivated FCS, at 37° in a 5% CO2 humidified atmosphere. After 24 hr, the culture medium was removed and fresh culture medium (supplemented with 10% heat-inactivated FCS and mycobacterial antigen) was added. In order to establish the optimal dose of antigen, cell-activation kinetic experiments were carried out over a 4-day time period using different protein concentrations of H37Rv M. tuberculosis culture supernatant. Con A mitogen (2 µg/ml) was used as a cell stimulation positive control. Cell stimulation was monitored at 24-hr intervals, by immunofluorescence determination of CD69 expression on CD4+ T cells.

Immunofluorescence staining of cell-surface markers and flow cytometry

Two-colour staining was performed on both PBMC and purified CD4+ T cells, by direct and indirect immunofluorescence, using mouse IgM anti-human CD57 and either FITC- or PE-labelled mAbs to human CD4, CD28, CD44, CD45RA, CD45RO, CD62L, TCR-αβ or TCR-γδ. Briefly, 2 × 105 cells were suspended in 20 µl of PBS containing 0.2% bovine serum albumin (BSA) and 0.2% sodium azide (Buffer 1), and incubated with the first-step mAb reagent for 30 min at room temperature. After incubation, the cells were washed twice with Buffer 1, and a second-step staining was performed with FITC- or Cy3-labelled anti-IgM antibodies. After 30 min of incubation, the cells were washed twice, fixed with 1% p-formaldehyde and analysed by flow cytometry.

All cells were analysed, for the expression of markers, on a FACScan flow cytometer (Becton Dickinson) using cellquest software, and 10 000 events were counted. To analyse the staining of cell-surface markers, the lymphocytes were first gated by their physical properties (forward and side scatter), then a second gate was drawn based on immunofluorescence characteristics of the gated cells, assessing fluorescence intensity by histograms. To analyse intracellular cytokine staining, the gates for positive and negative fluorescence of CD4+ T cells (forward scatter and fluorescence) were set manually based on the distribution of cells stained with isotypic controls alone. Data are presented as two-dimensional dot-plots, contour maps or histograms. Intensity of fluorescence staining is expressed as the mean fluorescence intensity (MFI). Control stains were performed using isotype-matched mAb of unrelated specificity labelled with FITC-, Cy3- or PE. Background staining was <1% and was subtracted from experimental values.

Flow cytometry analysis of intracellular cytokines

Four hours before mycobacterial protein antigen-activated CD4+ T-cell co-cultures ended, brefeldin-A was added (10 µg/ml). At the end of the incubation period, CD4+ T cells were harvested, washed with Buffer 1 and stained with murine IgM anti-human CD57 for 30 min. After washing, the cells were incubated with biotin-labelled rat anti-murine IgM for 30 min; after washing, the cells were incubated with CyChrome-labelled streptavidin for 30 min and washed with Buffer 1. T cells were fixed with 4% p-formaldehyde in PBS for 10 min at 4°. Subsequently, the cells were washed twice with PBS and permeabilized with saponin buffer (0.1% saponin, 0.01% pig IgG, 10 mm HEPES, 10% BSA in PBS), shaking gently for 10 min at room temperature. Afterwards, the cells were incubated with PE-labelled mAbs against human IL-4, and FITC-labelled mAbs against anti-human IFN-γ, using both isotype-matched immunoglobulin/FITC and immunoglobulin/PE controls.30 After 30 min, the cells were washed with PBS, fixed again with 1% p-formaldehyde and analysed immediately by flow cytometry, as discussed above.

Statistical analysis

Median values were compared, using the Mann–Whitney U rank sum test, by the sigma-plot8™ and sigma-stat2.03™ software. Values were considered to be statistically significant at a P-value of <0.05.

Results

CD4+ CD57+ T cells

First, we established the proportion of CD4+ CD57+ T cells in PBMC from healthy subjects and TB patients. The percentage of CD4+ cells was found to be similar, there was a twofold increase in the number of CD57+ cells in TB patients (Fig. 1a), but the frequency of CD4+ CD57+ T cells was 3.8 times higher in TB patients than in healthy control individuals (P < 0.001) (Fig. 1b).

Figure 1.

Figure 1

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Expression of cell-surface molecules in fresh peripheral blood mononuclear cells (PBMC), as analysed by flow cytometry on the FACScan, which was gated to include lymphocytes. Cells were stained with fluorescence-conjugated antibodies to CD4 and CD57 in a double-immunofluorescence assay, as described in the Materials and methods. (a) This figure is representative of one of 30 healthy controls and 30 tuberculosis (TB) patients studied; numbers in the upper right corner represent the percentage of lymphocytes in each quadrant. (b) Frequency of CD4+ CD57+ cells in PBMC from healthy controls and TB patients; the percentage of double-positive cells was plotted and the bars denote the median in each group. A P-value is indicated.

The surface phenotype of freshly isolated CD4+ CD57+ or CD4+ CD57− T cells from patients and healthy individuals was analysed. The vast majority of CD4+ CD57+ T cells exhibited high levels of CD45RO expression, suggesting the memory phenotype; cells from TB patients showed a 2.5-fold increased expression of CD45RO on CD4+ CD57+ T cells compared with CD4+ CD57− T cells. Similar results were obtained in cells from healthy control individuals; in both cases there was a statistical difference of P < 0.003 (Table 1).

Table 1.

Percentage of purified CD4+ T cells expressing surface markers

CD57+ cells CD57− cells
Controls Patients Controls Patients
CD45RA 19.9 ± 5.6 20.8 ± 7.1 29.2 ± 6.6 38.6 ± 8.2
CD45RO 79.0 ± 5.3* 81.1 ± 6.8† 40.9 ± 4.8* 31.6 ± 3.9†
CD62L 23.0 ± 5.2‡ 11.1 ± 3.3 50.7 ± 1.3‡ 27.1 ± 10.6
CD44 97.0 ± 1.1 94.6 ± 2.4 98.8 ± 0.7 91.8 ± 3.6
CD28 1.9 ± 0.4§ 8.0 ± 2.5 97.8 ± 0.6§ 89.9 ± 3.4
TCR-αβ 92.8 ± 3.1 66.8 ± 10 94.6 ± 0.4 67.3 ± 11.0
TCR-γδ 4.6 ± 0.4 12.4 ± 2.5** 5.0 ± 1.6 3.0 ± 1.5**
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Flow cytometry analysis was performed on freshly isolated CD4+ T cells, from peripheral blood mononuclear cells, by negative magnetic selection. Immediately, the cells were incubated with antibodies to CD57 and to other cell-surface markers by double immunofluorescence assay, as described in the Materials and methods. Results are expressed as the percentage of positive cells within the CD57+ or CD57− cell subpopulation. Numbers indicate mean values ± standard error, of at least five experimental samples, performed in triplicate, for each group. Significant differences were found in the comparison between CD57+ and CD57− cells from the control or patient group

*,†

P < 0.003

‡

P < 0.007

§,¶

P < 0.001

**

P < 0.01.

TCR, T-cell receptor.

The percentage of CD4+ CD57+ T cells expressing CD62L was twofold lower in healthy and TB patients in comparison to their CD4+ CD57− T cells; the difference was not statistically significant (Table 1). A similar observation was made when CD62L fluorescence intensity was measured on CD4+ CD57+ T cells (Fig. 2). There were no differences in CD44 expression on CD4+ T cells from patients or healthy controls, independently of their CD57 status (Table 1). CD28 expression showed a remarkable decrease on CD4+ CD57+ T cells in comparison with CD4+ CD57− T cells (P < 0.001) (Table 1).

Figure 2.

Figure 2

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Comparative histogram analysis of the CD62L fluorescence intensity on CD4+ CD57+ T cells (a) and CD4+ CD57− T cells (b). Purified CD4+ T cells were freshly isolated from peripheral blood mononuclear cells (PBMC) by negative magnetic selection and stained immediately with anti-CD57 and anti-CD62L fluorescent antibodies, as described in the Materials and methods. Lymphocytes were first gated by their physical properties (side-scatter and forward angle-scatter parameters), then only CD62L fluorescence was analysed on a second gate to include CD57+ T cells or CD57− T cells. The x-axis denotes the CD62L fluorescence intensity in tuberculosis patients (thick line) and in healthy controls (thin line); the y-axis indicates cell numbers. Data are representative of five assays for each group.

The percentage of TCR-αβ+ cells in TB patients was 0.7 times lower than in healthy control cells, independently of their CD57 expression. Nevertheless, in cells from TB patients, TCR-γδ expression on CD4+ CD57+ T cells was four times higher than on CD4+ CD57− T cells (P < 0.01) (Table 1).

In vitro stimulation of CD4+ T cells and their intracellular cytokine profile

Dose–response in vitro assays, using mycobacterial protein, showed that the maximal expression of CD69 (an activation marker)31 in co-cultured CD4+ T cells was induced by 3 µg of antigen (Fig. 3a). Under these culture conditions, we determined the percentage of cells from TB patients that expressed, intracellularly, IL-4 or IFN-γ (Fig. 3b). As shown in Table 2, there were 21-fold more IL-4+ cells in the CD57+ cell subpopulation. Similarly, there was an increase in the percentage of IFN-γ+ cells within the CD57+ cell subpopulation, although the increase was not as high as for IL-4+ cells.

Figure 3.

Figure 3

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Mycobacterial antigen-stimulated CD4+ T cells from tuberculosis (TB) patients. CD4+ T cells were isolated from peripheral blood mononuclear cells (PBMC), by negative magnetic selection, and co-cultured with monocytes (CD14+ adherent cells). (a) The cells were stimulated with specific soluble protein concentrations of mycobacterial antigen (H37Rv strain), after which the cells were harvested and stained with anti-CD69 fluorescent antibody. The black circles denote the mean value and bars indicate the standard error from three independent experiments. (b) Intracellular cytokines in antigen-stimulated CD4+ T cells from TB patients. Representative plot of two regions identified by positive or negative fluorescence to CD57 staining versus forward angle scatter. Region 1 (R1) includes CD4+CD57− T cells, whereas region 2 (R2) includes CD4+ CD57+ T cells. Arrows in histograms indicate the immunofluorescence of interferon-γ (IFN-γ) and interleukin-4 (IL-4) on the gated cells. Data are representative of five independent experiments.

Table 2.

Percentage of CD4+ T cells positive to intracellular cytokines

CD57+ cells CD57− cells
Mycobacterial antigen Con A mitogen Mycobacterial antigen Con A mitogen
IL-4+ 19.7 ± 3.2* 69.7 ± 4.5 0.9 ± 0.2* 22.5 ± 2.6
IFN-γ+ 24.7 ± 2.5† 75.2 ± 4.4 5.8 ± 0.8† 21.4 ± 3.6
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Flow cytometry analysis for CD4+ T cells co-cultured with autologous monocytes for 72 hr in the presence of mycobacterial antigen (3 µg/ml). Concanavalin A (Con A) (2 µg/ml) was used as the cell-activation control. At the end of the co-culture, CD4+ T cells were incubated with antibodies to CD57, interleukin-4 (IL-4) and interferon-γ (IFN-γ) for anaysis using three-colour immunofluorescence assays, as described in the Materials and methods. The percentage of positive cells was obtained from at least five samples for each group, and data are expressed as mean values ± standard error. Statistically significant differences were found in the comparison between CD57+ cells and CD57− cells from the group of tuberculosis patients

*,†

P < 0.001.

Discussion

The protective immune response to TB infection is maintained by effector/memory CD4+ T cells that are stimulated by the persistence of mycobacterial antigen.4–7 The presence of CD57+ T cells is associated with chronic antigenic stimulation.9–17 This is the first study to demonstrate a significant increase of the CD4+ CD57+ T-cell subset in the peripheral blood of patients infected with M. tuberculosis; we also found, similarly to d'Angeac et al.,24 who analysed healthy individuals, that the vast majority of these CD4+ CD57+ T cells showed a memory phenotype, as determined by their CD45RO expression.32 It is known that peripheral blood memory T cells from TB patients are activated during infection.33 Recently, Hengel et al.34 reported, in PBMC from non-treated TB/HIV patients, an increase in the proportion of IFN-γ+ CD45RA− CD62L− T cells after stimulation with mycobacterial antigen, in contrast to our observation which showed the CD45RA− CD45RO+ CD62L− phenotype in the great majority of non-antigen-stimulated CD4+ CD57+ T cells.

Interestingly, we observed a fourfold increase in γδ expression in CD57+ T cells. It has been long recognized that some γδ T cells have cytotoxic effector-cell properties35 and that some recognize non-peptide mycobacterial antigens.36 De Rosa et al.37 found two human γδ T-cell subsets: one with the CD28+ CD57− phenotype and the other with the CD28− CD57+ phenotype; this author suggests that the latter correspond to effector/memory cells. We do not know whether the γδ T cells isolated from our patients have recognition or cytotoxic properties, but the majority have the CD57+ phenotype.

Another interesting aspect is the diminution of the TCR-αβ expression on T cells isolated from patients with TB, which we believe is secondary to activation caused by chronic antigen exposure. Various authors have reported TCR-αβ down-regulation during T-cell activation.38–40

It has been reported that the interaction of CD28 with its natural ligand B7 (CD80/CD86) is critical for the normal T-cell activation process.41 Our results showed that the majority of CD4+ CD57+ T cells were negative for CD28, in accordance with results reported by others.42,43

As the protective immune response to M. tuberculosis is maintained predominantly by Th1 cells,6,34 we investigated the presence of intracellular cytokines in CD4+ CD57+ T cells after stimulation with mycobacterial antigen. Our stimulation experiments demonstrated a significant increase in the frequency of CD4+ CD57+ T cells that were positive to IFN-γ or IL-4. It would be important to study other regulatory cytokines to better define the real function of this cell subpopulation. Nevertheless, it has been recently shown that IL-4 induces the expression of CD1 on monocytes, thus enhancing the immune response to non-peptidic antigens.44,45

CD57 is a carbohydrate epitope19 with multiple functions: it is one of the proposed ligands for P- and L-selectin,21 a ligand for IL-622 and a ligand for nervous system proteoglycans.23 It is possible that the interaction of CD57 with P-selectin, expressed on activated endothelial cells, and with IL-6 (a result of chronic inflammation), favours the selective adhesion of a certain, as-yet unknown, CD4+ T-cell subpopulation with an activated memory phenotype and drives the immune-response cells to the area of tisular lesion. Altogether, these results suggest that the CD4+ CD57+ T cells could play a significant role in the immune response in TB patients.

Acknowledgments

Thanks are due to Rosa Nieto (INER) and Gisela Martinez (UNAM) for their technical assistance. Financial support: Consejo Nacional de Ciencia y Tecnología (CONACYT, 34814-M) and Dirección General de Asuntos de Personal Académico de la Universidad Nacional Autónoma de México (DGAPA-UNAM) proyecto PAPIIT (IN224598).

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