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Cell-mediated immune responses to influenza vaccination in Wegener's granulomatosis
  1. Albert Holvast1,
  2. Aalzen de Haan2,
  3. Sander van Assen3,
  4. Coen A Stegeman4,
  5. Minke G Huitema1,
  6. Anke Huckriede2,
  7. Cornelis A Benne5,
  8. Johanna Westra1,
  9. Abraham Palache6,
  10. Jan Wilschut2,
  11. Cees G M Kallenberg1,
  12. Marc Bijl1
  1. 1Department of Rheumatology and Clinical Immunology, University Medical Centre Groningen, University of Groningen, The Netherlands
  2. 2Department of Medical Microbiology, Molecular Virology Section, University Medical Centre Groningen, University of Groningen, The Netherlands
  3. 3Department of Internal Medicine, Division of Infectious Diseases, University Medical Centre Groningen, University of Groningen, The Netherlands
  4. 4Department of Nephrology, University Medical Centre Groningen, University of Groningen, The Netherlands
  5. 5Laboratory for Infectious Diseases, Groningen, the Netherlands
  6. 6Solvay Pharmaceuticals, Weesp, The Netherlands
  1. Correspondence to Dr A Holvast, Department of Rheumatology and Clinical Immunology, University Medical Centre Groningen, University of Groningen, PO Box 30.001, 9700 RB Groningen, The Netherlands; bertholvast{at}hotmail.com

Abstract

Background Both antibody and cell-mediated immune responses are involved in the defence against influenza. In Wegener's granulomatosis (WG), antibody responses to influenza vaccination appear to be similar to those in healthy controls, but cell-mediated responses have not been studied.

Objective To determine whether cell-mediated responses to influenza vaccination in WG vary from those in controls.

Methods Twenty-five patients with WG and healthy controls received subunit influenza vaccine. Peripheral blood mononuclear cells were obtained before and 1 month after vaccination. Cell-mediated responses to A/H1N1 and A/H3N2 were assessed using interferon γ (IFNγ) ELISpot and intracellular cytokine staining for IFNγ, tumour necrosis factor and interleukin 2.

Results Before vaccination, patients and controls showed similar recall responses to A/H1N1 and A/H3N2. After vaccination, patients and controls showed similar levels of increase in spot-forming cells against A/H1N1 and A/H3N2. By flow cytometry, upon vaccination, proportions of cytokine-producing CD4 T cells increased in patients and controls for A/H1N1 and A/H3N2.

Conclusions Cell-mediated responses to influenza vaccination in patients with WG are comparable to those in healthy controls.

Dutch Trials Register number NTR1130.

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Introduction

Wegener's granulomatosis (WG) is an autoimmune inflammatory disease affecting small and medium-sized vessels, which leads to granulomatous inflammation (particularly in the airways), systemic vasculitis and glomerulonephritis.1 Patients with WG are at risk for infections,2 and the median age of patients with WG is relatively high. Therefore, influenza vaccination should be considered in WG.

Influenza has a high incidence as approximately 5% of adults develop symptomatic influenza each year.3 Annual vaccination reduces the severity of influenza and related complications, and is recommended in immunocompromised patients.4 The immune response to influenza consists of both antibody and cell-mediated responses. In WG, antibody responses to influenza vaccination appear to be similar to those in healthy controls,5 6 but cell-mediated responses have not been studied. The latter are relevant as in certain groups, such as older people and young children, cell-mediated responses to influenza vaccination can be a marker of clinical protection, independent of antibody responses.7,,9

In WG, the level of antigen-specific T-cell responses to vaccination may be reduced because infection rates are increased and immunological disturbances have been described, such as skewing of the CD4 T-cell pool and dysfunctional regulatory T cells.10 11 In vivo generated cell-mediated immune responses, for example, after vaccination, have not been studied previously in WG. We performed an explorative study on cell-mediated responses to influenza vaccination in WG. This investigation was part of a study in which antibody responses to influenza vaccination were evaluated.6

Methods

Study population

Patients with WG with quiescent disease, Birmingham Vasculitis Activity Score (BVAS) <2 and age- and sex-matched healthy controls were included. Additional information is available as an online supplementary text file.

Study design

Patients with WG and controls received (from October to December 2005) subunit influenza vaccine (Influvac, 2005–2006; Solvay Pharmaceuticals, Weesp, The Netherlands), containing A/New Caledonia/20/99 (H1N1), A/NewYork/55/2004 (H3N2) and B/Hong Kong/330/2001. Before and 4 weeks after vaccination, peripheral blood mononuclear cells (PBMCs) were isolated. The study was approved by the institutional medical ethics committee, and informed consent was obtained from all participants.

ELISpot, ELISA and flow cytometry

Pre- and post-vaccination PBMC samples, from a patient with WG and a matched control, were simultaneously thawed and batch-processed. β-Propiolactone inactivated whole virus (WIV) of A/New Caledonia/20/99 (H1N1) and A/Hiroshima/52/2005 (H3N2) were used to stimulate PBMCs. A/Hiroshima/52/2005 is a very closely related antigenic variant of the vaccine strain A/NewYork/55/2004. For interferon γ (IFNγ) ELISpot assays, 2 × 105 PBMCs were added to each well, and incubated with WIV for 48 h. IFNγ ELISA was performed on supernatants from similar cultures. For flow cytometry assays, 1.0–1.5 × 106 PBMCs were cultured in culture medium alone and in the presence of Staphylococcal enterotoxin B (SEB; Sigma-Aldrich, Saint Louis, Missouri, USA), concanavalin A (Con A), WIV A/H1N1) and A/H3N2. Cells were incubated for 18 h, the final 16 h in the presence of brefeldin A (Sigma-Aldrich). Cells were labelled for CD3, CD4, CD8, CD69, IFNγ, tumour necrosis factor (TNF) and interleukin 2 (IL2). Complete information is available as an online supplementary text file.

Statistical analysis

Log-transformed before testing were ELISpot, ELISA, CD4/CD8 T-cell data on SEB and Con A, and CD8 T-cell data on A/H1N1. CD4 T-cell data on A/H1N1 were root-transformed before testing. All variables were tested using Student t test, except for previous influenza vaccination (Fisher's exact test) and CD4 T-cell responses against A/H3N2 (Mann–Whitney U tests and Wilcoxon signed rank test). For correlations, Pearson's and Spearman's correlation coefficients were used where appropriate. A two-sided p value <0.05 was considered statistically significant.

Results

Patient characteristics

Responses were evaluated in 25 patients with WG and in 25 controls; in one patient and one control cells were of insufficient viability. Characteristics are shown in table 1. During follow-up, one patient developed active disease (BVAS=5).

Table 1

Baseline characteristics and disease parameters

Cell-mediated immune responses

Activated (CD69) cytokine-producing T cells were quantified by flow cytometry. Upon SEB and Con A stimulation, patients with WG and controls showed similar frequencies of IFNγ-, TNF- and IL2-producing CD4 T cells and CD8 T cells (online supplementary figure 1). After assessing general responsiveness, we analysed influenza-specific, cell-mediated responses. By ELISpot, before vaccination, patients with WG had similar numbers of IFNγ spot-forming cells against A/H1N1 and A/H3N2 as compared with controls (figure 1A, p=0.632 and p=0.377, respectively). To assess functional capacity of these responding PBMCs, the total amount of IFNγ produced upon A/H1N1 and A/H3N2 stimulation was determined and again patients and controls did not differ (figure 1B).

Figure 1

Interferon γ (IFNγ) ELISpot and ELISA. (A) ELISpot of IFNγ-producing cells and (B) IFNγ ELISA, per 2 × 105 peripheral blood mononuclear cells (PBMCs), in patients with Wegener's granulomatosis (WG) and healthy controls (HC), in response to A/H1N1 and A/H3N2 stimulation before vaccination (T=0 days) and 4 weeks after vaccination (T=28 days). For ELISA, also responses to concanavalin A (Con A) are shown. Results are corrected for responses in unstimulated cultures from the same sample. Medians and interquartile ranges are shown. In ELISA, vaccination tended to increase IFNγ production in WG upon A/H1N1 stimulation (p=0.068).

By flow cytometry, prevaccination, the frequency of cytokine-producing CD4 T cells against A/H1N1 did not differ between patients and controls (figure 2A,B). For A/H3N2, only IFNγ-producing CD4 T cells could be detected, at similar frequencies in patients and controls (figure 2C). Within CD8 T cells, recall responses could only be detected for A/H1N1. Frequencies of IFNγ- and TNF-producing CD8 T cells were similar in patients and controls; IL2-producing CD8 T cells could not be detected (figure 2D).

Figure 2

CD4+ and CD8 T+ cell responses against A/H1N1 and A/H3N2. (A) Representative example of gating of activated (CD69) interferon γ (IFNγ)-producing CD4+ T cells, in a prevaccination sample of a patient with Wegener's granulomatosis (WG); unstimulated cells (left), stimulated with A/H1N1 (middle) and stimulated with A/H3N2 (right). Frequencies of cytokine-producing CD4+ T cells upon stimulation with (B) A/H1N1 and (C) A/H3N2 in patients with WG and healthy controls (HC), before vaccination (T=0 days) and 4 weeks after vaccination (T=28 days). (D) Frequencies of cytokine-producing CD8+ T cells upon stimulation with A/H1N1 in WG and HC, before vaccination. Results are corrected for responses in unstimulated cultures from the same sample. Medians and interquartile ranges are shown. IL, interleukin; ND, not detectable; TNF, tumour necrosis factor.

After vaccination, patients with WG and controls both showed a rise in IFNγ spot-forming cells against A/H1N1 and A/H3N2, to similar levels (figure 1A).

By flow cytometry, for A/H1N1, increases of frequencies of cytokine-producing CD4 T cells were modest and reached statistical significance only for IFNγ in patients and IL2 in controls (figure 2B). For A/H3N2, CD4 T-cell responses were detectable after vaccination, albeit at a lower level than those against A/H1N1. In patients, frequencies of TNF- and IL2-producing CD4 T cells increased; in controls, increases were seen for IFNγ, TNF and IL2 (figure 2C). For both A/H1N1 and A/H3N2, frequencies of cytokine-producing CD4 T cells were similar in patients and controls after vaccination. Vaccination did not induce a CD8 T-cell response (data not shown).

Correlations

Changes in numbers of spot-forming cells after vaccination correlated with changes in IFNγ+ CD4 T-cell frequencies for A/H1N1 and A/H3N2 in controls and for A/H3N2 in patients. CD4 T-cell responses did not correlate with antibody responses (online supplementary figure 2).

Influence of immunosuppressive agents and previous influenza vaccination

When patients with WG without immunosuppressive drugs (n=13) were compared with patients using any immunosuppressive drug (n=11), no differences were found (supplementary text file). Next, we compared patients who had received an influenza vaccination in the previous year (n=12) with patients who had not received vaccination in the previous year (n=12). In the previous year, the A/H1N1 vaccine strain was identical. Before and after vaccination, previously vaccinated patients and those not previously vaccinated showed no difference in cell-mediated immune responses (data not shown).

Discussion

In this study, we found no differences in cell-mediated immune responses to subunit influenza vaccination between patients with WG and controls. Cell-mediated recall responses to influenza were comparable between patients and controls. This is in accordance with previous studies, in which normal cytokine and proliferation responses to recall antigens in patients with WG have been reported.12 13 After vaccination, the number of spot-forming cells increased in both patients and controls to similar levels, and these appeared to be of comparable functional capacity. CD4 T-cell cytokine responses to influenza vaccination were modest, but measurable, and were observed for both A/H1N1 and A/H3N2 in patients and controls; again, levels remained similar in patients and controls. Responses were, at least largely, independent of previous vaccination status and the use of immunosuppressive drugs. Thus, functional capacity of the T-cell pool does not appear to be affected by the immunological disturbances in WG. This conclusion is restricted to patients with quiescent disease and patients receiving relatively mild immunosuppressive treatment. Active disease and/or use of more potent immunosuppressive agents, such as cyclophosphamide, may result in diminished cell-mediated immune responses.

In systemic lupus erythematosus, cell-mediated responses to influenza vaccination were reduced, which was associated with the use of prednisone and/or azathioprine.14 In WG, we did not observe such an effect, though prednisone and azathioprine were used in comparable doses. Possibly, other, disease-related, factors are involved in systemic lupus erythematosus. However, this comparison should be made cautiously, as the number of patients with WG was relatively small.

Frequencies of CD8 T cells were low and not detectable for A/H3N2. For A/H1N1, CD8 T-cell frequencies were recorded at similar levels in patients and controls. As expected with a subunit influenza vaccine, we found no change in cytokine-producing CD8 T cells after vaccination.15

We found no correlation between antibody responses and IFNγ spot-forming cells or CD4 cytokine-producing cells. This is in accordance with the observations of others.16 Therefore, cell-mediated and antibody responses appear to be independent measures of the response to influenza vaccination, as has also been suggested in several clinical studies.7,,9

This study has several limitations. First, the number of patients and controls studied was relatively small. Second, the use of immunosuppressive drugs was heterogeneous and relatively mild. Third, the proportion of participants who had received an influenza vaccination in the previous year was much higher among patients than controls. However, previous influenza vaccination did not seem to influence cell-mediated responses.

In conclusion, cell-mediated responses to subunit influenza vaccination were similar in patients with WG with quiescent disease and controls, despite the use of, relatively mild, immunosuppressive drugs in patients. Antibody responses to influenza vaccination are adequate, and no indications of detrimental effects upon disease activity have been reported. Therefore, influenza vaccination in patients with WG with quiescent disease can be recommended.

References

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Supplementary materials

Footnotes

  • Funding Unrestricted grants were received from Jan Kornelis de Cock Foundation, the Netherlands; Solvay Pharmaceuticals, Weesp, The Netherlands; Solvay Pharmaceuticals also, unrestrictedly, provided the vaccine.

  • Competing interests AP is employed by Solvay Pharmaceuticals. His contribution to this study and manuscript was unrestricted by Solvay Pharmaceuticals.

  • Ethics approval This study was conducted with the approval of the ethics committee of the University Medical Centre Groningen, Groningen, The Netherlands.

  • Provenance and peer review Not commissioned; externally peer reviewed.

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