Response of T-cell subpopulations to superantigen and recall antigen stimulation in systemic sclerosis.

BACKGROUND: There is great disagreement regarding which effector T-cells are responsible for the pathogenesis of systemic sclerosis. Further, the possible role of superantigens in modulating the T-cell phenotype responsible for the immunopathogenesis of this disease and the response of these patients to common recall antigens have not been adequately determined. AIMS: To investigate the T-cell subsets and activation markers in peripheral blood mononuclear cells of systemic sclerosis patients before and after stimulation with different bacterial superantigens and common recall antigens to better understand the immunopathogenesis of this disease.
MATERIALS AND METHODS: T-cells (CD3(+)) from 20 systemic sclerosis patients and 17 age-matched healthy controls were studied using flow cytometry for the expression of CD4, CD8, CD45RA, and CD45RO at baseline and upon stimulation with different superantigens and recall antigens. Patients were also tested for skin delayed hypersensitivity to common recall antigens.
RESULTS: The proportions of CD45RA(+) (naive) and CD45RO(+) (memory) CD4(+) T-cells were found to be significantly higher in patients than in controls upon stimulation with bacterial superantigens. However, T-cells from these patients responded weakly to recall antigen stimulation, indicating a loss of specific memory cells. This was further supported by the skin delayed hypersensitivity test in which 16 patients were found to be anergic.
CONCLUSIONS: Our findings suggest that both naïve (CD45RA(+)) and memory (CD45RO(+)) CD4(+) superantigen-reactive T-cells are effector T-cells that may modulate the pathogenic autoantibody response in systemic sclerosis. Accumulation of these cells in these patients may result in increased risk of relapses and resistance to therapy.

Introduction

Scleroderma is a systemic autoimmune disease with a heterogeneous and complex phenotype that encompasses several distinct clinical subtypes. The disease has an estimated prevalence of 276 cases per million adults in the United States.[12] There are a few reports on the epidemiology of scleroderma from India that suggest that the disease is not uncommon and that, due to increase in life expectancy, its prevalence is also increasing in our country. A review of Indian literature published in 1970 reported 23 cases of scleroderma over a span of 25 years.[3] Another study found that 98 cases of scleroderma have been reported over a 16-year period.[4]

One major type of scleroderma is systemic sclerosis (SSc), which is a generalized disorder of small arteries, microvessels, and connective tissue. It is a disease of unknown origin, with the highest incidence occurring between 45 to 55 years of age;[5] the frequency is three to eight times higher in females.[6] Several studies have demonstrated that the extent of skin involvement directly correlates with internal organ involvement and prognosis in SSc patients.[7-9]

A wide range of B- and T-cell abnormalities have been described in SSc, including antinuclear antibodies, immune complexes, decreased number of circulating T-lymphocytes, and increased CD4+/CD8+ ratio (due to decreased number of CD8+ T-cells).[1011] Previous reports on T-lymphocyte subpopulations in SSc are partially conflicting.[12-15] Further, microbial superantigens (sAgs) have been identified as one of the possible candidates responsible for causing or aggravating autoimmune disease.[1617] Autoreactive T- and B-cells circulate in the peripheral blood of otherwise healthy patients;[1819] sAgs could stimulate them both locally and systemically since they do not discriminate between autoreactive and naïve lymphocytes,[20] thereby breaking the delicate balance of tolerance.

Persistent in vivo exposure to sAgs/environmental toxins in the periphery can lead to significant expansions of committed T-cell subpopulations, followed by clonal anergy or depletion that is reflected as decreased response to recall antigen (rAg) stimulation.[21] Because the relative number of variable-region genes is limited in humans, a given sAg is capable of interacting with a large fraction (5–30%) of T-cells expressing the target T-cell receptor. By this process, a large proportion of the patient's immune system could be activated, with reactivity to a wide repertoire of antigenic determinants, shortly after infection with an sAg-carrying agent. The ability to stimulate polyclonal B (IgG) as well as T-cell responses raises the possibility of a role for sAgs in the induction of various autoimmune diseases. Although sAgs are yet to be directly implicated in human autoimmune disease, there is growing evidence to suggest their involvement.[22-26]

In India, skin infections by Staphylococcus and Streptococcus species are common due to the tropical climate. Immunologic changes induced by the sAgs of these microbes may alter the incidence and progression of autoimmune diseases like SSc. However, there is no study on the risk of various staphylococcal and streptococcal sAgs in SSc. These antigens can modify the clinical expression of SSc as well as its response to treatment. Therefore, we initially wanted to investigate the T-cell subsets – CD4+ and CD8+ cells – and the expression of CD45RA+ and CD45RO+ on these T-cell subsets in peripheral blood mononuclear cells (PBMCs) of SSc patients before and after stimulation with different bacterial sAgs; we also wanted to study the response of these patients to common rAgs in vivo and in vitro to better understand the immunopathogenesis of this disease.

Materials and Methods

Patients

Twenty patients with SSc, attending the Department of Dermatology between January 2009 and June 2010, were enrolled in the study. All of the patients satisfied the American College of Rheumatology's preliminary criteria for classification as definite SSc[27] and protocol items specific for SSc were determined by established convention. All the patients included were either fresh cases or were not on steroids/immunosuppressive drugs/other therapeutic agents since the last 6 months. The patients were subjected to routine baseline clinical laboratory investigations. Stool examination of all the patients was done to look for ova/cysts of parasites to understand the role of these infections in causing immunological changes in SSc. Seventeen age-matched healthy controls (HC) subjects were also enrolled for comparison. The study was approved by the ethics committee of the hospital and written informed consent was obtained from the patients before enrollment in the study.

Peripheral blood mononuclear cell preparation

Peripheral venous blood (10 ml) was collected aseptically from patients and HC in EDTA vacutainers and used for isolation of PBMCs according to a modification of the method of Boyum.[28] Briefly, the samples were diluted 1:1 with Hank's balanced salt solution (HBSS), layered over HiSep™ LSM (HiMedia Laboratories Pvt. Ltd., Mumbai, India), and centrifuged for 30 min at 500 g at room temperature. The cloudy layer at the interface was carefully aspirated and washed using HBSS. The viability of the cells was measured by trypan blue exclusion assay and was always found to be above 95%. The PBMCs obtained were resuspended in complete medium [RPMI 1640 media, supplemented with 10% FBS, 100 IU/ml of penicillin, 100 μg/ml of streptomycin and 2 mM L-glutamine (HiMedia Laboratories Pvt. Ltd.)] at a final concentration of 1 × 106 cells/ml.

Stimulation of PBMCs

PBMCs (1 × 106 cells/ml) in complete medium were seeded in the wells of a 12-well cell culture plate (Nunc) in the presence of different concentrations of various sAgs and rAgs. The superantigens used were streptococcal pyrogenic exotoxin A (SPEA; 1-100 ng/ml) and staphylococcal enterotoxin B (SEB; 1–100 ng/ml) (Toxin Technology, USA). The recall antigens used were Candida antigen (CA; 10–100 ng/ml) (prepared by a method published earlier)[29] and purified protein derivative (PPD; 5 TU) (Span Diagnostics Ltd., India). The mitogen phytohemagglutinin-M (PHAM) (Sigma-Aldrich, Delhi, India) was used at 10 μg/ml concentration, which was determined by titration previously. All stimulations were done in triplicate and the cells were incubated at 37°C in humid air containing 5% CO2 for 72 h.

Monoclonal antibodies

Monoclonal antibodies (MAbs), CD3, CD4, CD8, CD45RA, and CD45RO conjugated to peridine chlorophyll (PerCP), fluorescein isothiocyanate (FITC), phycoerythrin (PE), and allophycocyanin (APC) were used for immunostaining and analysis by fluorescence-activated cell sorting (FACS). All the antibodies, including the corresponding isotype controls, were purchased from BD Biosciences, Gurgaon, India.

Staining of PBMCs and FACS

PBMCs were harvested after 72 h of incubation and washed three times with wash buffer (0.5% BSA + 0.1% NaN3 in PBS) before staining with combinations of different antibodies conjugated to PerCP, FITC, PE, and APC. Staining was done on ice for 30–60 min in the dark, after which the cells were washed thrice with wash buffer and fixed with 200 μl of cold 1% paraformaldehyde in PBS. FACS scanning was performed within 18 h with FACSCalibur™ (BD Biosciences). About 20000 to 30000 events were collected per condition and data were analyzed using WinMDI® 2.9 software. Appropriate isotype control antibodies were included in all the analysis.

Skin delayed hypersensitivity testing

Before initiating immunosuppressive therapy, 5 TU (tuberculin units) PPD, 5–25 Lf (flocculation units) of tetanus toxoid (TT), and 0.5 ml of diphtheria, pertussis, and tetanus (DPT) (Serum Institute of India Ltd.) were injected intradermally in the lateral one-third of the left forearm of patients. The reaction was read by measuring the diameter of the palpable, raised, hardened area across the forearm (perpendicular to the long axis) in millimeters after 48 h. A score of ≤2 mm diameter to a single antigen was taken as anergy and a result of >2 mm was taken as positive. Glycerine was used on the other arm as control.

Statistical analysis

Data is presented as the mean value±SE. Proportions of lymphocyte subpopulations were compared using the unpaired Student's t-test. Statistical analysis was performed using SigmaStat® 2.03 software.

Results

Clinical profile of SSc patients

Twenty SSc patients (sixteen females and four males) with age varying from 14 years to 55 years (mean age 34.6±3.86 years) were studied. The duration of the disease varied from 2 months to 14 years (mean duration 3.3±1.6 years). Patients most commonly presented with Raynaud phenomenon (95%), skin sclerosis (90%), and pigmentation (90%). Other common clinical manifestations were contracture of fingers (65%), digital ulceration (60%), dyspnea (55%), mouth opening restriction (50%), joint complaints (40%), and dysphagia (30%). The Rodnan skin score varied from 9 to 51 (mean score 27.4±4.31). Seventeen age-matched HC (eleven females and six males) were also studied for comparison. The profile and clinical characteristics of the subjects are given in Table 1.

Table 1

Profile and clinical characteristics of the SSc patients in the study

The memory/naive T-cells in circulation of SSc patients are predominantly CD4+ and sAg reactive

We studied T-cell phenotype from SSc patients (n=20) and HC (n=17) before and after stimulation with SPEA and SEB, the two most common sAgs encountered in skin infections. The concentrations of sAgs resulting in optimal stimulations were determined in preliminary experiments and were found to be 100 ng/ml for both the sAgs (data not shown). Most of the SSc patients displayed a considerably higher CD4+/CD8+ ratio than that observed in HCs [Figure 1]. Intriguingly, CD4+/CD8+ ratio was found to be notably lower in four of the SSc patients who were found to be positive for helminthic infections (Ascaris lumbricoides).

Figure 1

Difference in mean percentages of different subsets of T-cells (CD3+) between SSc patients (n=20) and HCs (n=17). SSc patients have more activated CD4+ T-cells compared to HCs. (a) The mean percentage of CD4+, CD8+, and CD45RA+/ RO+ T-cells from SSc patients and HCs before stimulation; (b) mean percentages after stimulation with SPEA (100 ng/ml); and (c) mean percentages after stimulation with SEB (100 ng/ml). *P<.05, **P<.001, ***P<.0001 compared with healthy controls

We also observed significant differences in the proportions of CD45RA+ and CD45RO+ T-cells between SSc patients and HCs [Figure 1]. Upon superantigenic challenge, CD4+ T-cells of SSc patients showed marked increase in activation/expression of CD45RA+ and RO+ phenotype. This was not seen in the CD8+ T-cell population of the same patients [Figure 2].

Figure 2

Expression of CD45RA+ and CD45RO+ activation markers on CD4+ and CD8+ T-cells in unstimulated (US) and stimulated (SPEA 100 ng/ ml and SEB 100 ng/ml) cells of SSc patients. (a) The mean percentages of CD45RA+/RO+ on CD4+ T-cells, and (b) the mean percentages of CD45RA+/ RO+ on CD8+ T-cells. *P<.05, **P<.001, ***P<.0001 compared with CD8+ T-cells

T-lymphocyte response to rAg stimulation in SSc

Recall antigens (CA and PPD) and the mitogen PHAM were also tested in an in vitro stimulation assay. Optimal concentrations were 100 ng/ml for CA, 5 TU for PPD, and 10 μg/ml for PHAM. No statistically significant differences were observed in the percentages of CD4+ T-cells between SSc patients and HCs upon rAg stimulation, but as regard to CD8+ T-cells, significantly lower activation was observed in SSc patients than in HCs. Also, significantly lower expression of activation markers CD45RA+ and CD45RO+ on T-cells was observed in SSc patients than in HCs. A representative flow cytometric analysis of these observations is given in Figure 3. The response of T-lymphocytes from SSc patients and HCs to PHAM was almost similar.

Figure 3

Flow cytometric analysis of PBMCs of a representative individual from each group (SSc and HC) stimulated with 100 ng/ml of CA in vitro. The figure depicts the percentage of T-cells (CD3+) that are positive for CD4 and CD8, and activation markers CD45RA (naive) and CD45RO (memory). Higher activation of CD8+ (P<.001), CD45RA+ (P<.0001), and CD45RO+ (P<.001) T-cells was observed in HCs than in SSc patients

Skin delayed hypersensitivity reaction

All the SSc patients were tested intradermally with PPD (5 TU), TT (5–25 Lf), and DPT (0.5 ml) for skin delayed hypersensitivity reaction to these antigens. Twelve of the patients were found to be anergic (diameter ≤2 mm) to all the three antigens tested and four were anergic to PPD and DPT only. There were four patients who showed positivity (diameter >2 mm) to all the antigens tested, while four showed positivity to TT only [Table 2]. All the HC tested with these antigens showed a diameter of >2 mm.

Table 2

Skin delayed hypersensitivity reaction of SSc patients to common rAgs

Discussion

Systemic sclerosis is predominantly characterized by T-cell activation and deposition of collagen, leading to vascular damage and progressive tissue fibrosis.[27] The alterations of T-cell behavior of SSc patients was explicitly demonstrated by challenging them with sAgs in vitro. The potential role of sAgs in triggering T-cell activation was confirmed/supported by a higher expression of CD4+ T-cells in patients with SSc. SPEA and SEB being T-cell stimulatory protein molecules produced primarily by Streptococcus and Staphylococcus species, respectively,[30] can therefore nonspecifically activate the host T-cells to permit the expression of acute disease.[17] These sAgs interact with a specific Vβ region of the TCR, stimulating a large fraction of peripheral circulating T-cells (≥10%).[31] High expression of activation markers CD45RA (naïve) and CD45RO (memory) on CD4+ T-cells of SSc patients, as compared to HC, suggests that both these cell populations might be involved in autoimmune pathogenesis of SSc, and that effector T-cells involved in SSc presumably exist in the CD4+ T-cell subpopulation. These findings are consistent with reports showing an increased CD4/CD8 ratio in SSc patients when compared with HC.[3233] Although the role of sAgs in SSc has not been subjected to extensive research, our study suggests a role for sAgs as a possible stimulus for the increased numbers of CD4+ T-cells in these patients. There are reports where evidence of involvement of these antigens in other human autoimmune diseases has been demonstrated.[2526]

We, however, observed a significant increase in the percentages of cytotoxic CD3+CD8+ T-cells in four of the SSc patients, which was conspicuous and probably related to the chronic/persistent A lumbricoides infection observed in these patients (data not shown). Decreased CD4+ and increased CD8+ counts, with T-cell activation, has been associated with chronic helminthic infection in an earlier study.[34] Depletion of CD4+ cells could represent the net result of several coexisting events occurring at different levels in these patients.

CD4+ T-cells of SSc patients responded differently to common rAgs (CA and PPD) challenge in vitro. Their proliferative response was not as high as was observed with sAgs challenge. Also, CD8+ T-cells and activation markers of SSc responded weakly to these antigens, as compared to HCs. The poor response of CD45RO+ to rAgs perhaps reflects the dearth of functional memory cells which leads to ‘anergy’ in these patients. On assessing the vitality of the T-cell response of these patients by skin delayed hypersensitivity test, using PPD, TT, and DPT, we observed anergy (diameter ≤2 mm) in 16 of 20 patients, indicating a broad lack of T-cell responsiveness that supported our in vitro findings. There is now wide agreement about the presence of depressed cutaneous cell-mediated immunity in rheumatic disorders.[3536] This may be because of immunological suppression and nonspecific activation of immune cells in these patients, which in turn may lead to loss of specific effector memory T-cells. These anergic patients can have exacerbation of lesions due to sAgs/environmental toxin trigger. Such a paradoxical suppression of peripheral immune responses, along with a high degree of clinical anergy, may contribute to susceptibility to and persistence of proinflammatory responsiveness in the disease through sAg/environmental antigen challenge.

The observation of Raynaud phenomenon and skin sclerosis (>90% cases) and digital ulceration (60% cases) in these SSc patients may be indicative of the strength of the patients’ immune response to contain the diseases. SSc patients with high levels of anergy suggest a direct relationship between the anergy and the underlying immunopathogenic activity of the disease. The functional recovery of CD8/memory pool of T-cells and the cytokine response after specific therapy may indicate a reversal of the chronicity of the disease.

Thus, it may be reasonable to conclude (although the underlying mechanism in the regulation and maintenance of effector T-cells need to be further elucidated) that both naïve (CD45RA+) and memory (CD45RO+) CD4+ T-cells play the role of effector T-cells in SSc, possibly modulating the pathogenic autoimmune response in these patients. The accumulation of sAg-reactive T-cells may result in an increased chance of relapses/resistance to therapy in these patients and, therefore, steroid/immunosuppressive therapy given to these patients should be carefully monitored.