SEARCH

SEARCH BY CITATION

Abstract

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

Objective

Expanded populations of CD4+CD28− T cells with a cytotoxic phenotype have been repeatedly reported in patients with granulomatosis with polyangiitis (Wegener's) (GPA). In healthy individuals expansion of this T cell population follows cytomegalovirus (CMV) infection. We undertook this study to investigate whether CMV infection may be responsible for driving the expansion of CD4+CD28− T cells in GPA patients and how this might relate to clinical features.

Methods

Forty-eight GPA patients and 38 age-matched healthy donors were included in the study. CMV-specific IgG in serum was detected by enzyme-linked immunosorbent assay. Flow cytometric analysis was used to study T cell populations and phenotype. The presence of CMV in renal biopsy tissue from GPA patients was investigated by immunohistochemistry and polymerase chain reaction (PCR). Clinical information was obtained from patient records.

Results

Populations of CD4+CD28− T cells were only expanded in CMV-seropositive GPA patients and controls. In CMV-seropositive GPA patients we observed negative correlations between the percentages of CD4+CD28− T cells and both the percentage of naive T cells and the glomerular filtration rate at presentation. There was a significant association between the percentage of CD4+CD28− T cells and risk of infection and mortality. CMV could not be detected in renal tissue by PCR or immunohistochemistry. CMV seropositivity itself was not a risk factor for infection in a cohort of 182 patients with antineutrophil cytoplasmic antibody–associated vasculitis who had been recruited into clinical trials performed by the European Vasculitis Study Group.

Conclusion

The expansion of CD4+CD28− T cells in GPA patients is associated with CMV infection and leads to a reduction in the number of naive T cells in peripheral blood. Patients with expanded CD4+CD28− T cells have significantly increased mortality and risk of infection.

Antineutrophil cytoplasmic antibody–associated vasculitis (AAV), comprising granulomatosis with polyangiitis (Wegener's) (GPA) and microscopic polyangiitis (MPA), is a chronic inflammatory disease associated with organ damage affecting the lungs and kidneys. AAV is associated with autoantibodies against proteinase 3 and myeloperoxidase, which are thought to be directly pathogenic, but the potential role of the cellular immune response in AAV is less certain (1). Abnormalities of the T cell repertoire have been consistently reported in AAV, and a recurrent finding is an increased percentage of CD4+ T cells that have lost expression of CD28 (2–5). CD28 is a costimulatory molecule that interacts with B7-1 and B7-2 on antigen-presenting cells, and increased frequencies of CD4+CD28− T cells have been reported in several inflammatory conditions including AAV (6, 7). CD4+CD28− cells are unusual within the CD4+ cell repertoire in that they have cytotoxic activity and show strong expression of perforin and granzyme B (8). CD4+CD28− cells are strongly expanded in cytomegalovirus (CMV)–seropositive individuals, and it has been demonstrated that the expansion of these cells in rheumatoid arthritis is associated with CMV infection (9).

Here we show that the expansion of CD4+CD28− cells that has been previously demonstrated in patients with GPA is found only in CMV-seropositive individuals. Patients with increased numbers of CD4+CD28− T cells have a reduction in the number of naive T cells within the peripheral circulation. Moreover, the expansion was also correlated with the severity of renal disease and associated with an increased risk of infection.

PATIENTS AND METHODS

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

Patients.

Forty-eight patients with GPA were recruited from the vasculitis clinic at University Hospitals Birmingham National Health Service Foundation Trust. The patients had stable remission of disease with low-dose immunosuppression (maximum prednisolone dose 5 mg/day with either mycophenolate mofetil or azathioprine). The median duration of disease was 4.5 years (interquartile range [IQR] 1.9–6.6 years) for CMV-seronegative patients and 4.2 years (IQR 1.8–7.4 years) for CMV-seropositive patients (P not significant).

Twenty-five CMV-seropositive AAV patients (17 with GPA, 8 with MPA) were additionally studied at disease presentation, and clinical data were collected prospectively for 12 months. Patients' immunosuppressive therapy was tailored to the severity of their disease. All patients received remission induction therapy with high-dose steroids. Twenty-two patients received cyclophosphamide (either daily oral or pulsed intravenous), 1 patient received methotrexate, and 2 patients with relapsing disease and previous cyclophosphamide exposure received mycophenolate mofetil. The median cyclophosphamide exposure was 5.2 grams (IQR 3–10.6 grams). Ten patients received an additional 4 infliximab infusions (5 mg/kg body weight per infusion).

Retrospective clinical information regarding episodes of infection was obtained by examination of hospital medical records from the time of diagnosis until the date of the study. All significant documented infections (requiring antibiotic therapy and/or admission to hospital) were included, although some infections managed elsewhere and not reported to the team managing patients in the vasculitis clinic may have been missed. Thirty-eight age-matched healthy control blood donors were recruited from local volunteer cohorts. There was no significant difference in the proportion of CMV-seropositive and CMV-seronegative donors in the healthy control and GPA patient groups. Demographic details for vasculitis patients and healthy donors are shown in Table 1.

Table 1. Demographic features of the patients with disease in remission (at time of sampling), healthy controls, and AAV patients with active disease (at time of recruitment)*
 Healthy controls (n = 38)GPA patients with disease in remission (n = 48)AAV patients with active disease (n = 25)
  • *

    There were no significant differences between healthy controls and patients with granulomatosis with polyangiitis (Wegener's) (GPA). AAV = antineutrophil cytoplasmic antibody–associated vasculitis; IQR = interquartile range; CMV = cytomegalovirus.

Age, median (IQR) years57 (41–77)64 (47–74)67 (59–74)
No. CMV positive/no. CMV negative23/1531/1725/0
No. of men/no. of women13/2525/2314/11

Serum samples were also available from AAV patients recruited into clinical trials performed by the European Vasculitis Study Group (EUVAS) (10–13), and these were used for CMV IgG serotyping. Extensive clinical data were available from this group. All blood and serum donors gave informed consent, and all work was approved by local research ethics committees. The research was conducted in accordance with the Declaration of Helsinki. CMV-specific IgG was detected in the serum by enzyme-linked immunosorbent assay (BioCheck, Inc.) according to the manufacturer's instructions.

Cell staining with antibodies and flow cytometry.

Peripheral blood mononuclear cells (PBMCs) were isolated from heparinized blood samples by Ficoll-Hypaque density-gradient centrifugation (Lymphoprep; Axis-Shield) and cryopreserved in fetal calf serum (FCS) containing 10% DMSO. After washing with phosphate buffered saline (PBS) containing 0.5% bovine serum albumin and 2 mM EDTA, antibodies against surface markers were added and the cells were incubated for 15 minutes on ice. We used a 7-color panel. Phycoerythrin (PE)–Cy7–conjugated anti-CD4 (eBioscience) and AmCyan-conjugated anti-CD8 (BD Biosciences) were used to identify T cell subsets. Phenotypic analysis of these subsets was done using allophycocyanin–Alexa Fluor 750–conjugated anti-CD27 (eBioscience), fluorescein isothiocyanate–conjugated anti-CD28 (BD Biosciences), PE-conjugated anti-CD57 (Southern Biotechnology), Alexa Fluor 700–conjugated anti-CD45RA (BioLegend), and energy-coupled dye (ECD)–conjugated anti-CD45RO (Beckman Coulter). After another wash, samples were analyzed using a Becton Dickinson LSR II flow cytometer and FACSDiva (BD Biosciences) or FlowJo (Tree Star) software.

Cell sorting and 5,6-carboxyfluorescein succinimidyl ester (CFSE) proliferation assays.

To assess the CMV reactivity of the CD4+CD28− T cell populations expanded in CMV-seropositive donors, we sorted CD4+ T cells from fresh PBMCs into CD28− and CD28+ populations on a MoFLow (Beckman Coulter). The sorted cells were then labeled with 0.5 μM CFSE (Molecular Probes/Invitrogen) in PBS for 5 minutes at 37°C followed by 5 minutes of incubation in ice-cold RPMI 1640 containing 10% FCS on ice. The cells were then washed and resuspended in RPMI 1640 plus 10% FCS. For the proliferation assay, 3–5 × 104 sorted T cells were cultured in the presence of 1.5 × 105 antigen-pulsed autologous irradiated PBMCs for 5 days. Cells were stimulated with CMV lysate, staphylococcal enterotoxin B (SEB), tuberculin purified protein derivative (PPD), medium alone, or irradiated PBMCs only. Proliferative capacity of the cells was assessed on day 5 by flow cytometry using an LSR II flow cytometer and FACSDiva software and calculated as the percentage of proliferated cells minus background proliferation detected following stimulation with irradiated PBMCs only (background).

Immunohistochemical staining for CMV antigen.

Formalin-fixed paraffin-embedded tissue sections (4 μm) of renal biopsy samples (n = 21) were dried at 37°C overnight and for 30 minutes at room temperature prior to being dewaxed and hydrated. Sections were incubated in 0.1% trypsin in PBS for 15 minutes at 37°C and then washed, and endogenous peroxidases were blocked by immersion for 10 minutes in 1% H2O2 in methanol. Again, samples were washed in running water and then washed in PBS for 5 minutes, followed by incubation with mouse anti-CMV antibody (M0854; Dako) at 1:200 dilution for 60 minutes. After another wash in PBS the sections were incubated in Envision (Dako) for 30 minutes, placed in 3,3′-diaminobenzidine for 10 minutes, washed, counterstained, dehydrated, cleared in xylene, and mounted.

Quantitative polymerase chain reaction (qPCR) for CMV.

Total genomic DNA was isolated from renal biopsy tissues (n = 21) and analyzed with qPCR to determine the presence of CMV DNA. For this assay, sections were cut from paraffin-embedded renal biopsy samples, and DNA was isolated from these sections using the Nucleospin Tissue XS Kit (Macherey Nagel) according to the manufacturer's protocol.

The DNA samples were spectrophotometrically quantified (NanoDrop ND-1000; NanoDrop Technologies), and the quality was determined using a multiplex PCR consisting of 3 primer sets that formed 150-bp, 255-bp, and 343-bp products. This was followed by agarose (1%) gel electrophoresis. DNA was accepted as “good quality” when all 3 products were formed.

Quantitative PCR for the detection of viral DNA was carried out in a total volume of 25 μl per reaction containing AmpliTaq Gold PCR Master Mix (Roche Molecular Systems), TaqMan probe (FAM-5′-TGCATGAAGGTCTTTGCCCAGTACATTCT-3′-TAMRA; 10 pmoles), sense primer (5′-AGCGGCCTCTGATAACCA-3′; 25 pmoles), antisense primer (5′-ACTAGGAGAGCAGACTCTCAGAGGAT-3′; 25 pmoles), and 10 μl sample DNA (60–1,700 ng). The qPCR program consisted of an initial denaturation and enzyme activation step (15 minutes at 95°C), followed by 50 PCR amplification cycles consisting of denaturation (15 minutes at 95°C), annealing (15 minutes at 63°C), and extension (15 minutes at 72°C), followed by a final extension step (5 minutes at 72°C) forming a 127-bp PCR product size.

Statistical analysis.

Statistical analysis was performed using SPSS, version 17.0 software. The Kolmogorov-Smirnov test was used to confirm if data conformed to a normal distribution. When possible, non-normally distributed data were transformed to achieve a normal distribution. Paired or unpaired t-tests, Mann-Whitney U tests, or Wilcoxon signed rank tests were used to examine differences between 2 groups of data. Analysis of variance or Friedman's tests were used to examine differences between 3 groups of data. Pearson's product-moment correlation coefficients or Spearman's rank correlation coefficients were used to investigate significant correlations between data sets. Data are presented as the median (IQR) or mean ± SD. The effects of factors on survival times were investigated using Kaplan-Meier survival curves or Cox regression analysis. Cumulative cyclophosphamide and prednisolone exposures were entered into the Cox model as segmented time-dependent covariates. Hazard ratios (HRs) and 95% confidence intervals (95% CIs) are provided. P values less than 0.05 were considered significant.

RESULTS

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

Expansion of CD4+CD28− T cells in CMV-seropositive individuals.

In an initial analysis we determined the percentage and absolute counts of CD4+CD28− T cells within the peripheral blood of GPA patients and healthy controls. The CD4+CD28− subset comprised a median 7.6% (IQR 1.5–34) of the CD4+ T cell pool in GPA patients compared with a median 7.5% (IQR 1.5–27) in healthy control subjects. When these values were correlated with CMV serostatus, it was clear that expansion was only seen in CMV-seropositive individuals. Specifically, the median (IQR) was 0.8% (0.4–1.7) and 19% (8–50) in the CMV-seronegative and CMV-seropositive patient groups, respectively, compared with 1.4% (0.8–1.9) and 22% (12–42) in seronegative and seropositive controls, respectively (Figure 1A). Similarly, despite a reduction in absolute CD4+ and CD4+CD28+ T cell counts in CMV-seropositive GPA patients, an expanded population of CD4+CD28− T cells was observed (Figure 1B). Statistical analysis thus revealed that there was no difference in the size of the CD4+CD28− T cell pool between GPA patients and healthy controls in relation to CMV serostatus.

thumbnail image

Figure 1. Flow cytometric analysis of peripheral blood mononuclear cells in healthy controls and patients with granulomatosis with polyangiitis (Wegener's) (GPA). Data are shown as box plots. Each box represents the 25th to 75th percentiles (interquartile range [IQR]). Lines outside the boxes represent the 10th and the 90th percentiles. Lines inside the boxes represent the median. Circles indicate outliers. Asterisks indicate extreme outliers more than 3 IQRs from the end of the box. A, Frequencies of CD4+CD28− T cells are significantly expanded in cytomegalovirus (CMV)–seropositive donors in both groups studied. B, CMV-seropositive GPA patients have lower CD4+ and CD4+CD28+ cell counts than do CMV-seronegative GPA patients. CD4+CD28− T cell counts are higher in CMV-seropositive GPA patients than in CMV-seronegative GPA patients. C, Shown are the peripheral blood lymphocyte count (shaded boxes) and CD4+CD28− T cell counts (open boxes) in GPA patients at time of presentation with active disease, through to remission at 6–12 months, and finally during long-term remission (3–5 years). The lymphocyte count decreases during treatment but the CD4+CD28− cell count remains stable. D, The percentage of CD4+CD28− T cells increases over time.

Download figure to PowerPoint

Increased percentage of CD4+CD28− T cells following treatment with immunosuppression.

To determine how the number and percentage of CD4+CD28− T cells was related to the clinical stage of the disease, we performed a prospective analysis of T cell phenotype in 12 of the 25 patients who were followed up sequentially from presentation with active disease, through to remission at 6–12 months, and finally during long-term remission (3–5 years). As anticipated, the absolute lymphocyte count fell during the treatment phase from a median (IQR) of 1.2 × 109/liter (0.8–1.7) at presentation to 0.8 × 109/liter (0.6–1.3) at 6–12 months and 0.7 × 109/liter (0.6–1.1) during long-term remission. This was associated with a decrease in the total CD4+ lymphocyte count over the same period of time (Figure 1C). The absolute number of CD4+CD28− T cells was unchanged between presentation, 6–12 months, and 3–5 years in GPA patients (Figure 1C), but there was a significant increase in the percentage of CD4+CD28− T cells over time (Figure 1D). This suggests that CD4+CD28− T cells are relatively resistant to the lymphocyte-depleting action of cyclophosphamide.

Association of the phenotype of CD4+CD28− T cells with CMV serostatus.

The phenotype of the CD4+CD28− T cell pool was then determined by study of the surface expression of a range of markers associated with different differentiation profiles. In CMV-seropositive donors the cells exhibited a predominant CD27−CD45RA−CD45RO+CD57+ profile, in keeping with previous reports (14) (Figure 2).

thumbnail image

Figure 2. Expression of CD57 (A), CD27 (B), CD45RO (C), and CD45RA (D) on CD4+CD28− T cells in CMV-seropositive and CMV-seronegative GPA patients and healthy controls. Data are shown as box plots. Each box represents the 25th to 75th percentiles. Lines outside the boxes represent the 10th and the 90th percentiles. Lines inside the boxes represent the median. Circles indicate outliers. Asterisks indicate extreme outliers more than 3 IQRs from the end of the box. CD4+CD28− T cells show a markedly different phenotype in CMV-seronegative and CMV-seropositive donors in both subject groups. See Figure 1 for definitions.

Download figure to PowerPoint

In contrast, the phenotype of CD4+CD28− cells in the CMV-seronegative controls and GPA patients was markedly different, with increased expression of CD27 and CD45RA, but reduced staining with antibodies to CD45RO and CD57 (Figure 2). Interestingly, after correction for CMV serostatus, no significant differences were observed in the phenotype of CD4+CD28− T cells between GPA patients and healthy controls.

Proliferation of CD4+CD28− T cells in response to CMV antigen.

The presence of CD4+CD28− T cells was strongly associated with CMV seropositivity and a differentiated phenotype of the expanded cells. To determine the CMV reactivity of these cells we investigated the proliferative responses of CD28− and CD28+ CD4+ cells to CMV lysate in 4 GPA patients with disease in remission (Figure 3A). CD4+ T cells were sorted from fresh PBMCs into CD28− and CD28+ populations, subsequently labeled with CFSE, and stimulated with antigen-pulsed irradiated PBMCs. Following 5 days of incubation the response of the CD4+ T cells was assessed using flow cytometry. Although the proportion of cells proliferating in response to CMV varied between donors (Figure 3B), a significantly higher proportion of CD28− than CD28+ cells responded to CMV in each donor. Responses against a non–CMV-related antigen (PPD) were only detected in the CD4+CD28+ T cell population. Interestingly, the proliferative response to CMV in the CD4+CD28− cells from each donor was very similar to the response to SEB, whereas in the CD4+CD28+ cells the response to SEB was much greater than the response to CMV. This raises the possibility that the remaining CD4+CD28− cells were anergic rather than specific to other antigens.

thumbnail image

Figure 3. CD4+CD28− T cells respond to CMV antigen. A, Representative example of flow cytometry data. Sorted, 5,6-carboxyfluorescein succinimidyl ester (CFSE)–labeled CD4+CD28− and CD4+CD28+ T cells were stimulated with antigen-pulsed irradiated peripheral blood mononuclear cells (PBMCs) or staphylococcal enterotoxin B (SEB) or tuberculin purified protein derivative (PPD) as controls, and proliferation was assessed on day 5 following stimulation. Proliferating cells are seen in the left-hand quadrants showing CFSE dilution. B, Summary of the proliferation data on 4 GPA patients studied in this way. The proportion of cells responding to CMV lysate varied between patients, although a greater response was always seen within the CD28− population than within the CD28+ population. See Figure 1 for other definitions.

Download figure to PowerPoint

Association of CMV seropositivity with a decrease in the percentage of naive T cells in GPA patients.

Previous studies have shown that GPA patients often exhibit a profile of increased numbers of memory T cells together with a reduced naive T cell pool (2). To determine how this pattern was influenced by CMV seropositivity we used CD27 and CD45RA to define the 4 major CD4+ memory T cell populations: naive (CD27+CD45RA+) (Figure 4A), central memory (CD27+CD45RA−) (Figure 4B), effector memory (CD27−CD45RA−) (Figure 4C), and CD45RA+ “revertant memory” (CD27−CD45RA+) (Figure 4D) (15).

thumbnail image

Figure 4. Proportions of memory cell subsets determined in the CD4+ T cell population in healthy controls and GPA patients in relation to CMV serostatus. Data are shown as box plots. Each box represents the 25th to 75th percentiles. Lines outside the boxes represent the 10th and the 90th percentiles. Lines inside the boxes represent the median. Circles indicate outliers. Asterisks indicate extreme outliers more than 3 IQRs from the end of the box. Shown are proportions of CD27+CD45RA+ naive cells (A), CD27+CD45RA− central memory cells (B), CD27−CD45RA− effector memory cells (C), and CD27−CD45RA+ “revertant memory” cells (D). CMV seropositivity leads to a reduction in the number of naive T cells in GPA patients, while effector memory T cell populations expand significantly. See Figure 1 for definitions.

Download figure to PowerPoint

Our results revealed no overall difference in the percentage of memory cells and naive cells in GPA patients compared with controls. However, CMV seropositivity was strongly associated with a reduced pool of naive cells in GPA patients, such that naive cells represented a median of only 10% (IQR 5–23) of the CD4+ pool in CMV-seropositive patients compared with 45% (IQR 35–61) in the CMV-seronegative group (Figure 4A). CMV infection was also associated with a marked increase in the percentage of effector memory and CD45RA+ “revertant memory” cells (Figures 4C and D) both in controls and in GPA patients. In control subjects CMV seropositivity was additionally associated with a reduced percentage of central memory cells, which was not observed in GPA patients (Figure 4B).

Strong correlation of the expansion of CD4+CD28− T cells with a reduction in naive T cells.

Although the expansion of CD4+CD28− T cells was strongly associated with CMV serostatus, a marked variation was observed in the percentage of such cells in the CMV-seropositive group, with a range between 0.9% and 46% of the CD4+ T cell pool. To establish whether this factor was important in determining the observed reduction in the naive T cell pool in CMV-seropositive donors, we plotted the percentage of CD4+CD28− T cells against the percentage of naive T cells. Importantly, a strong negative correlation was observed between the percentage of CD4+CD28− T cells and the percentage of naive CD4+ T cells in the GPA patients (Spearman's rank correlation coefficient −0.58, P = 9 × 10−6). After transformation of the data to achieve a normal distribution, a strong negative linear relationship was observed between the percentages of naive and CD4+CD28− cells (Figure 5A) in CMV-seropositive GPA patients (Pearson's product-moment correlation coefficient −0.7, P = 1 × 10−5). No relationship was observed between the percentages of naive and CD4+CD28− T cells in the CMV-seronegative patient group. Similar significant correlations between the percentages of CD4+CD28− T cells and naive T cells were observed in CMV-seropositive, but not in CMV-seronegative, healthy volunteer donors (data not shown).

thumbnail image

Figure 5. A, Correlation between CD4+CD28− T cells and naive T cells in CMV-seropositive and CMV-seronegative GPA patients. The best-fit line is shown for CMV-seropositive patients only. A significant linear relationship was observed between naive T cells and CD4+CD28− T cells in CMV-seropositive GPA patients (Pearson's product-moment correlation coefficient −0.7, P = 1 × 10−5), but not in CMV-seronegative GPA patients. To achieve a normal distribution, the percentages of both T cell populations were statistically transformed. B, Kaplan-Meier plot of infection-free survival from diagnosis. CMV-seropositive GPA patients were divided into tertiles based on the percentage of CD4+CD28− T cells. The time to first infection was determined for the tertile with the greatest expansion of CD4+CD28− T cells (group 2 [G2]; thick solid line), for the combined 2 tertiles with least expansion of CD4+CD28− T cells (group 1 [G1]; dotted line), and for the CMV-seronegative GPA patients (thin solid line). There was a significant difference in infection-free survival across the 3 groups (P = 0.041 by log rank test). When tested independently, there was no difference between CMV-seronegative and CMV-seropositive group 1 GPA patients. CMV-seropositive group 2 patients had a significantly reduced infection-free survival time compared with either of the other groups. The table below the survival plot shows the number of patients at risk for each time point of first infection. See Figure 1 for definitions.

Download figure to PowerPoint

Association of elevated numbers of CD4+CD28− T cells with impaired renal function, but without CMV replication in the kidney.

Since we had observed a marked variation in the percentage of CD4+CD28− T cells in GPA patients, we then went on to determine how this value might relate to clinical characteristics. There was a correlation between the percentage of CD4+CD28− T cells in CMV-seropositive GPA patients and their renal function (determined by estimated glomerular filtration rate [GFR] at presentation) (r = −0.37, P = 0.05). Since the percentage of CD4+CD28− cells had increased since the time of diagnosis, we confirmed this correlation in 25 patients who had also had phenotyping studies performed at the time of diagnosis, concomitant with the estimated GFR measurement (r = −0.39, P = 0.05).

Based on these findings we investigated whether the impaired renal function in these patients was driven by CMV replication in the kidney or whether it might reflect an indirect consequence of the expansion in CD4+CD28− T cells. Renal biopsy material was available from 21 CMV-seropositive patients, and tissue sections were analyzed for the presence of CMV by immunohistochemistry and PCR. Interestingly, CMV antigen was not detected in any of those samples, which shows that tissue damage secondary to viral replication is unlikely to explain the renal impairment observed in patients with an expanded CMV-specific immune response.

Increased risk of infection in patients with high numbers of CD4+CD28− T cells during remission.

There is now considerable interest in how the CMV-specific immune response might impair immunity to heterologous infections in CMV-seropositive individuals. In particular, increased mortality rates have been seen in CMV-seropositive elderly patients, and this is strongly associated with expansion of the T cell memory pool (16). We therefore investigated whether GPA patients with an expanded CD4+CD28− T cell pool were at increased risk of infection. Cox proportional hazards survival analysis demonstrated that both an increased percentage of CD4+CD28− T cells (HR 1.7 [95% CI 1.1–2.8], P = 0.027 for normal distribution–transformed percentage of CD4+CD28− T cells) and the absolute number of CD4+CD28− T cells (HR 3.0 per 105 cells/ml [95% CI 1.5–6.4], P = 0.003) were associated with an increased risk of infection. In contrast, elevated percentages (but not absolute counts) of naive T cells in CMV-seropositive GPA were associated with a 75% reduction in the risk of infection (HR 0.234 [95% CI 0.07–0.74], P = 0.014 for normal distribution–transformed percentage of naive T cells).

CMV-seropositive GPA patients were divided into tertiles based on the percentage of CD4+CD28− T cells. The time to first infection was determined for the tertile with the greatest expansion of CD4+CD28− T cells and for the combined 2 tertiles with least expansions of CD4+CD28− T cells, and these times to first infection were compared with that for the CMV-seronegative cohort (Figure 5B). No difference was observed in the time to infection between the CMV-seronegative group and the group of patients with least expansions of CD4+CD28− T cells. However, patients with expanded populations of CD4+CD28− T cells demonstrated an increased risk of infection, such that 50% of this group had had an episode of infection at 5 months compared with 13 months in the CMV-seronegative group. There was no association between age at diagnosis, renal function, cyclophosphamide exposure, and risk of infection in the CMV-seropositive patients.

Association of the percentage of CD4+CD28− T cells at presentation with increased risk of infection and increased mortality.

Since we had shown that expansion of CD4+CD28− T cells was associated with established risk factors for mortality in AAV (worse renal function and infection) (17), we investigated the effect of CD4+CD28− T cell expansion on outcomes in the 25 CMV-seropositive AAV patients who were followed up prospectively for 12 months from diagnosis. During the 1-year followup, 12 patients had an episode of infection and 6 patients died. Cox regression survival analysis showed an increased risk of mortality (HR 1.087 [95% CI 1.004–1.176], P = 0.039) and an increased risk of infection (HR 1.076 [95% CI 1.007–1.15], P = 0.03) for each 1% increase in percentage of CD4+CD28− T cells. The absolute count of CD4+CD28− T cells was associated with an increased risk of infection (HR 1.086 per 105 cells/ml [95% CI 1.004–1.175], P = 0.039) but not mortality in CMV-seropositive GPA patients. The risks of mortality and infection were independent of age, renal function, cumulative steroid exposure, cumulative cyclophosphamide exposure, or use of infliximab. No episode of infection in this cohort coincided with leukopenia.

Lack of association of CMV seropositivity with impaired clinical outcome in patients with vasculitis.

To determine if CMV serostatus itself is independently associated with an increased risk of infection in GPA patients, we studied serum samples available from 182 AAV patients who had participated in EUVAS trials. Within this group 103 patients had a diagnosis of GPA, while 62 had MPA and 17 had a diagnosis of renal limited vasculitis. One hundred twenty-two patients were CMV seropositive, while 60 were defined as CMV seronegative. The median time to first infection was 17.5 months and 18.6 months in CMV-seropositive and CMV-seronegative patients, respectively, with infection rates per patient-year of 0.3 and 0.2, respectively. Neither of these differences was statistically significant. CMV serostatus also was not correlated with either diagnosis or renal function at presentation. These data indicate that CMV infection by itself is not associated with impaired clinical outcome in patients with vasculitis, but it is the magnitude of the immune response to CMV that determines the relative infection risk.

DISCUSSION

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

Previous studies have demonstrated an expansion of CD4+CD28− T cells in the peripheral blood and granulomatous lesions of GPA patients (8). In healthy donors this population is found almost exclusively in individuals who have acquired latent CMV infection (16) and can account for up to 20% of the CD4+ T cell pool. CD4+CD28− T cells have been reported to develop shortly after cessation of viral replication in primary CMV infection of renal transplant recipients (18). Herein, we show for the first time that the expansion of CD4+CD28− T cells seen in peripheral blood of GPA patients is closely associated with CMV infection and, importantly, does not differ from that seen in age-matched healthy control subjects.

CD4+CD28− T cells have a cytotoxic phenotype and typically express CD57, granzyme B, and perforin (18). The presence of telomere shortening and restricted Vβ T cell receptor gene usage has been taken to reflect high proliferative capacity secondary to repeated viral replication (19–21). In addition to their cytotoxic potential, it has been suggested recently that these cells might have regulatory function (22).

CD4+CD28− T cells have been reported in several autoimmune diseases including GPA, where they have been associated with more severe disease and reported to be prolific producers of the inflammatory cytokines tumor necrosis factor α and interferon-γ (7, 8, 23, 24). CD4+CD28− T cells have been found in granulomatous lesions in GPA patients and are thought to contribute to disease progression, either directly by maintaining the inflammatory response or as a result of bystander activation (20). In our study, the percentage of CD4+CD28− T cells at diagnosis correlated with renal function at presentation, although the pathogenesis of this association is unclear, since we could not detect CMV directly in kidney biopsy samples from GPA patients. However, in patients with other autoimmune diseases such as rheumatoid arthritis and multiple sclerosis it has been demonstrated that these cells do not respond to autoantigen but proliferate in response to stimulation with CMV antigen, suggesting that they may play an important role in amplification of tissue damage rather than in the initial break in immune tolerance (25). Although we did not investigate responses to autoantigen, we also demonstrated significant proliferation of CD4+CD28− T cells in response to CMV lysate, confirming the CMV specificity of these cells.

In addition to the expansion of the CD4+CD28− T cells, we found a dramatic decrease in the frequency of naive T cells in this patient group, and the 2 measurements correlated in a linear manner. The mechanism behind this correlation is uncertain, but it may reflect the expansion of CD4+CD28− T cells taking up “space” in the peripheral immune system. Naive cells are responsible for the generation of immune responses to newly encountered antigen, and the physiologic significance of this association is revealed by the increased frequency of infection in this patient group.

We have therefore shown that the expansion of CD4+CD28− T cells is driven by CMV infection and that the expansion of these cells is associated with more severe renal disease and an increased rate of infection, both of which are independent prognostic factors associated with increased mortality in AAV patients (17). In the group of 25 patients studied from the time of presentation, the percentage of CD4+CD28− T cells appeared to be an independent risk factor for mortality. This raises the question of whether it is possible to intervene with antiviral drugs to suppress CMV replication and reduce the expansion of these cells, and thereby improve clinical outcomes for patients.

To determine if CMV infection can, by itself, act as a factor modulating the clinical outcome of patients, we studied a cohort of 182 EUVAS patients with AAV. These patients showed no increased risk of infection or mortality in association with CMV, which suggests that the degree of expansion of the CD4+CD28− T cell subset is the critical factor that determines the clinical significance of CMV infection.

In summary, we have demonstrated that the expansion of CD4+CD28− T cells in GPA depends on the presence of CMV infection, and that the magnitude of this increase is correlated with the severity of the renal disease. This expansion reduces the number of naive T cells in the peripheral circulation and increases the infection rate. These observations reveal that the magnitude of the CMV-specific immune response is an important determinant of clinical outcomes in GPA, and they suggest that antiviral medication may be considered as a potentially valuable mechanism to improve the clinical outcome of this disease.

AUTHOR CONTRIBUTIONS

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Harper had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Study conception and design. Morgan, Pachnio, Savage, Moss, Harper.

Acquisition of data. Morgan, Pachnio, Begum, Roberts, Rasmussen, Neil, Bajema, Harper.

Analysis and interpretation of data. Morgan, Pachnio, Moss, Harper.

Acknowledgements

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

The EUVAS was supported by the European Union (contracts BMH1-CT93-1078, CIPDT94-0307, BMH4-CT97-2328, and ERBIC20-CT97-0019).

REFERENCES

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES
  • 1
    Morgan MD, Harper L, Williams J, Savage C. Anti-neutrophil cytoplasm-associated glomerulonephritis. J Am Soc Nephrol 2006; 17: 122434.
  • 2
    Abdulahad WH, van der Geld YM, Stegeman CA, Kallenberg CG. Persistent expansion of CD4+ effector memory T cells in Wegener's granulomatosis. Kidney Int 2006; 70: 93847.
  • 3
    Popa ER, Stegeman CA, Bos NA, Kallenberg CG, Tervaert JW. Differential B- and T-cell activation in Wegener's granulomatosis. J Allergy Clin Immunol 1999; 103: 88594.
  • 4
    Schlesier M, Kaspar T, Gutfleisch J, Wolff-Vorbeck G, Peter H. Activated CD4+ and CD8+ T-cell subsets in Wegener's granulomatosis. Rheumatol Int 1995; 14: 2139.
  • 5
    Giscombe R, Nityanand S, Lewin N, Grunewald J, Lefvert A. Expanded T cell populations in patients with Wegener's granulomatosis: characteristics and correlates with disease activity. J Clin Immunol 1998; 18: 40413.
  • 6
    Moosig F, Csernok E, Wang G, Gross W. Costimulatory molecules in Wegener's granulomatosis (WG): lack of expression of CD28 and preferential up-regulation of its ligands B7-1 (CD80) and B7-2 (CD86) on T cells. Clin Exp Immunol 1998; 114: 1138.
  • 7
    Lamprecht P, Mueller A, Gross WL. CD28- T cells display features of effector memory T cells in Wegener's granulomatosis [letter]. Kidney Int 2004; 65: 1113.
  • 8
    Komocsi A, Lamprecht P, Csernok E, Mueller A, Holl-Ulrich K, Seitzer U, et al. Peripheral blood and granuloma CD4+CD28 T-cells are a major source of interferon γ and tumour necrosis factor α in Wegener's granulomatosis. Am J Pathol 2002; 160: 171724.
  • 9
    Hooper M, Kallas EG, Coffin D, Campbell D, Evans TG, Looney RJ. Cytomegalovirus seropositivity is associated with the expansion of CD4+CD28− and CD8+CD28− T cells in rheumatoid arthritis. J Rheumatol 1999; 26: 14527.
  • 10
    Jayne DR, Gaskin G, Rasmussen N, Abramowicz D, Ferrario F, Guillevin L, et al. Randomized trial of plasma exchange or high-dosage methylprednisolone as adjunctive therapy for severe renal vasculitis. J Am Soc Nephrol 2007; 18: 21808.
  • 11
    De Groot K, Harper L, Jayne DR, Flores Suarez LF, Gregorini G, Gross WL, et al. Pulse versus daily oral cyclophosphamide for induction of remission in antineutrophil cytoplasmic antibody-associated vasculitis: a randomized trial. Ann Intern Med 2009; 150: 67080.
  • 12
    Jayne D, Rasmussen N, Andrassy K, Bacon P, Tervaert JW, Dadoniene J, et al. A randomized trial of maintenance therapy for vasculitis associated with antineutrophil cytoplasmic autoantibodies. N Engl J Med 2003; 349: 3644.
  • 13
    De Groot K, Rasmussen N, Bacon PA, Tervaert JW, Feighery C, Gregorini G, et al, for the European Vasculitis Study Group. Randomized trial of cyclophosphamide versus methotrexate for induction of remission in early systemic antineutrophil cytoplasmic antibody–associated vasculitis. Arthritis Rheum 2005; 52: 24619.
  • 14
    Moins-Teisserenc H, Busson M, Scieux C, Bajzik V, Cayuela JM, Clave E, et al. Patterns of cytomegalovirus reactivation are associated with distinct evolutive profiles of immune reconstitution after allogeneic hematopoietic stem cell transplantation. J Infect Dis 2008; 198: 81826.
  • 15
    Serriari N, Gondois-Rey F, Guillaume Y, Remmerswaal E, Pastor S, Messal N, et al. B and T lymphocyte attenuator is highly expressed on CMV-specific T cells during infection and regulates their function. J Immunol 2010; 185: 31408.
  • 16
    Pourgheysari B, Khan N, Best D, Bruton R, Nayak L, Moss PA. The cytomegalovirus-specific CD4+ T-cell response expands with age and markedly alters the CD4+ T-cell repertoire. J Virol 2007; 81: 775965.
  • 17
    Little MA, Nightingale P, Verburgh CA, Hauser T, De Groot K, Savage C, et al. Early mortality in systemic vasculitis: relative contribution of adverse events and active vasculitis. Ann Rheum Dis 2009; 69: 103643.
  • 18
    Van Leeuwen EM, Remmerswaal EB, Vossen MT, Rowshani AT, Wertheim-van Dillen PM, van Lier RA, et al. Emergence of a CD4+CD28 granzyme B+, cytomegalovirus-specific T cell subset after recovery of primary cytomegalovirus infection. J Immunol 2004; 173: 183441.
  • 19
    Vogt S, Iking-Konert C, Hug F, Andrassy K, Hansch GM. Shortening of telomeres: evidence for replicative senescence of T cells derived from patients with Wegener's granulomatosis. Kidney Int 2003; 63: 214451.
  • 20
    Thewissen M, Somers V, Venken K, Linsen L, van Paassen P, Geusens P, et al. Analyses of immunosenescent markers in patients with autoimmune disease. Clin Immunol 2007; 123: 20918.
  • 21
    Kato M, Matsuguchi T, Ono Y, Hattori R, Ohshima S, Yoshikai Y. Characterization of CD28CD4+ T cells in living kidney transplant patients with long-term allograft acceptance. Hum Immunol 2001; 62: 133545.
  • 22
    Tovar-Salazar A, Patterson-Bartlett J, Jesser R, Weinberg A. Regulatory function of cytomegalovirus-specific CD4+CD27 CD28 T cells. Virology 2009; 398: 15867.
  • 23
    Lamprecht P, Bruhl H, Erdmann A, Holl-Ulrich K, Csernok E, Seitzer U, et al. Differences in CCR5 expression on peripheral blood CD4+CD28 T-cells and in granulomatous lesions between localized and generalized Wegener's granulomatosis. Clin Immunol 2003; 108: 17.
  • 24
    Lamprecht P, Moosig F, Csernok E, Seitzer U, Schnabel A, Mueller A, et al. CD28 negative T-cells are enriched in granulomatous lesions of the respiratory tract in Wegener's granulomatosis. Thorax 2001; 56: 7517.
  • 25
    Thewissen M, Somers V, Hellings N, Fraussen J, Damoiseaux J, Stinissen P. CD4+CD28null T cells in autoimmune disease: pathogenic features and decreased susceptibility to immunoregulation. J Immunol 2007; 179: 651423.