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. 2014 Sep 4;178(1):40–47. doi: 10.1111/cei.12390

Rituximab impairs immunoglobulin (Ig)M and IgG (subclass) responses after influenza vaccination in rheumatoid arthritis patients

J Westra *, S van Assen †, K R Wilting ‡, J Land *, G Horst *, A de Haan ‡, M Bijl §
PMCID: PMC4360192  PMID: 24889761

Abstract

Rituximab (RTX) treatment in rheumatoid arthritis (RA) patients severely hampers humoral response after influenza vaccination as determined by haemagglutination inhibition assay (HI). It is not known whether HI reflects both immunoglobulin (Ig)M and IgG (subclass) influenza response, and whether IgM antibodies contribute to the low rate of influenza infection seen in RA patients. Twenty RA patients on methotrexate (MTX), 23 on RTX and 28 healthy controls (HC) received trivalent influenza subunit vaccination. Before and 28 days after vaccination, H1N1- and H3N2-specific antibodies were measured by HI and by IgM and IgG (subclass) enzyme-linked immunosorbent assay (ELISA). B cell activating factor (BAFF) levels were determined in serum samples before vaccination. Vaccination induced a significant increase of IgM and IgG (IgG1 and IgG3) antibodies against both strains in the HC and MTX groups (all P < 0·01), but not in the RTX group. HI correlated significantly in all cases with IgG (IgG1) but not with IgM. In RTX late patients (RTX treatment 6–10 months before vaccination), IgG (IgG1 and IgG3) response to vaccination was restored, but not IgM response. BAFF levels were significantly increased in RA-RTX patients and correlated with total IgG levels. Haemagglutination inhibition assay, used as gold standard, detects primarily IgG (IgG1) responses. IgM- and IgG influenza-specific antibodies increase after vaccination in HC and RA patients except in patients on RTX treatment. BAFF levels are increased in both early and late RTX-treated patients, but do not correlate with an influenza-specific antibody response.

Keywords: IgG antibodies, IgM antibodies, influenza, rheumatoid arthritis, rituximab

Introduction

Rheumatoid arthritis (RA) patients are susceptible to many types of infection, especially after biological treatment 1–5. One of these infections is caused by the influenza virus. Influenza consists of three RNA subtypes (A, B and C), of which influenza A is the most frequently occurring one, and is subtyped based on the surface glycoproteins haemagglutinin (HA) and neuramidase (NA) 6.

Vaccination in RA patients on therapy seems to be safe 7–9 and effective even in patients on disease-modifying anti-rheumatic drugs (DMARDs), prednisone or anti-tumour necrosis factor (TNF) therapy 10. However, rituximab (RTX) therapy hampers the humoral vaccination response to influenza measured by haemagglutination inhibition assay (HI) 11,12. RTX is a chimeric monoclonal antibody directed against the CD20 cell surface molecule that is located on B cells. It causes B cell depletion and thereby significantly reduces the humoral response to vaccination. Despite this reduced response rate, recurrent infection is relatively low in patients on RTX compared to anti-TNF treatment 13. This might be due to a (relatively) intact cellular immunity or an adequate immunoglobulin (Ig)M response. Indeed, reports on the effects of cellular response to influenza vaccination are limited, but until now no significant influence on cellular response has been seen 14. Most studies determine levels of influenza-specific antibodies using the HI. Titres ≥40 are considered protective in healthy adults 15. It is not known whether HI reflects both IgM and IgG responses. Moreover, it is not known which IgG subclasses contribute to the IgG response to influenza. Measuring IgM antibodies is important to monitor early response to vaccination, and early response indicates the capacity of repopulating naive B cells to respond to the vaccine antigen. IgG1 and IgG3 are important immunoglobulins for complement fixation and binding to Fc receptors, which could play a role in antibody-dependent cellular toxicity.

One of the factors that controls B cell survival, B cell maturation and immunoglobulin class (IgG, IgA and IgE)-switching is B cell-activating factor (BAFF), also named B lymphocyte stimulator (BLys). It has been reported that BAFF levels increase after RTX treatment in RA patients 16. In a recent paper it was shown that RA synovial fibroblasts can produce high levels of BAFF that induce class-switching to IgA and IgG in IgD+ B cells 17. Chen et al. showed that soluble BAFF enhanced the humoral immune response by elevating B lymphocyte activity of secretion of immunoglobulins in chickens that were immunized 18. Whether BAFF levels are related to response to vaccination in RTX-treated patients is not known.

In this study we examined IgM and IgG(subclass) antibody response against influenza subunits measured with an enzyme-linked immunosorbent assay (ELISA), and compared the results to traditional HI. In addition, we analysed the relation between BAFF levels and influenza vaccination response in RA patients on MTX and RTX therapy.

Materials and methods

Patients

Twenty-eight healthy controls (HC) and 43 RA patients were included. All patients fulfilled the American College of Rheumatology clinical classification criteria for RA 19. Twenty patients were on MTX (in two patients combined with other DMARDs) and 23 patients on RTX (treatment of 11 patients 4–8 weeks before vaccination and of 12 patients 6–10 months before vaccination). Data were retrieved from a previous study 12. Patient characteristics are shown in Table 1. The mean age of RTX and MTX patients did not differ, but was higher in both groups of RA patients compared to HC (P = 0·004). Patients in the RTX group had lower numbers of B cells than patients in the MTX and HC groups (both P < 0·001) as a result of RTX treatment. The RTX patients were recruited in four participating Dutch University Medical Centres 12. HC and patients on MTX (including some on additional DMARDs) were recruited from the Groningen University Medical Centre. Exclusion criteria were: (i) lack of informed consent, (ii) age under 18, (iii) malignancy, (iv) pregnancy and (v) known allergy to or former severe reaction following vaccination with trivalent influenza subunit vaccine. The study was approved by the ethics committees of all participating centres.

Table 1.

Baseline characteristics of rheumatoid arthritis (RA) patients, treated with rituximab (RTX) or methotrexate (MTX) and healthy controls (HC)

RA-RTX (n = 23) RA-MTX (n = 20) Healthy controls (n = 28)
Age, mean ± s.d. years 55·5 ± 7·6 57·1 ± −6·7 45·2 ± 11·3#
Gender, female (%) 16 (70) 11 (55) 22 (78·6)
Previous vaccination (%) 12 (52) 10 (50) 20 (71·4)
Duration RA median years 13·8 8·7
MTX dosage, median (range) 17·5 (10–25) n = 10 16·3 (10–25) n.a.
Prednisone dosage, median (range) mg/day 8·75 (3·8–40) n = 15 0 (0–0) n.a.
Other DMARDs n(%) 1 (4) 2 (10)
IgG, g/l, median (range) 9·6 (3·4–16·7)* 11·5 (8–14·9) 11·6 (7·6–18·4)
 IgG1 5·9 (2·3–10·4) 6·6 (4·9–9·8) 6·3 (3·7–8·6)
 IgG2 2·2 (0·7–5·5)* 2·6 (1·3–4·8) 2·9 (1–8·3)
 IgG3 0·4 (0·1–0·9) 0·6 (0·2–1·2) 0·4 (0·1–1·4)
 IgG4 0·2 (0–1·1) 1 (0–1·8) 0·7 (0–1·8)
IgA, g/l, median (range) 2·2 (0·4–4) 2·1 (0·8–5·3) 1·8 (0·9–5·6)
IgM, g/l, median (range) 0·98 (0·2–2·3) 1 (0·5–4·3) 0·8 (0·3–2)
Interval before vaccination (%)
4–8 weeks after rituximab 11 (48)
6–10 months after rituximab 12 (52)
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#P < 0·01 compared to RA-RTX and RA-MTX. *P = 0·05 compared to HC. DMARDS = disease-modifying anti-rheumatic drugs; Ig = immunoglobulin; n.a. = not applicable; s.d. = standard deviation.

Vaccine

Trivalent subunit influenza vaccine (Solvay Pharmaceuticals, Weesp, the Netherlands) was used, and contained the following strains: A/Wisconsin/67/2005 (H3N2)-like strain, A/Solomon Islands/3/2006 (H1N1)-like strain and B/Malaysia/2506/2004-like strain. Immediately before and 28 ± 3 days after vaccination blood was drawn from patients and controls, and after centrifugation was stored at −20 C until use.

Methods

Antibody levels against all three strains were measured before and 28 days after vaccination using HI. HI was performed with guinea pig erythrocytes following standard procedures 20, and results have been reported previously 12.

Specific anti-influenza antibodies, both IgM and IgG (subclasses), were determined by an ELISA in all samples before and after vaccination. In short, microtitre plates were coated with 1 μg/ml subunit of A/H1N1 or A/H3N2 and with F(ab′)2 goat anti-IgG (Jackson Immunoresearch, Newmarket, UK) for IgG standard curve or with monoclonal anti-human IgM (clone MBII; Sigma-Aldrich, Zwijndrecht, the Netherlands) for IgM standard curve. Serum samples in multiple dilutions were added and IgG, IgM, IgG1, IgG3 and IgG4 standard curves were set up. Detection was performed with horseradish peroxidase (HRP)-labelled mouse anti-human IgG (clone JDC-10), mouse anti-human IgM (clone SA-DA4), mouse anti-human IgG3 (clone HP6050) and mouse anti-human IgG4 (clone HP6025), all from Southern Biotech (Birmingham, AL, USA), and mouse anti-human IgG1 (clone MH161-1, Fitzgerald, North Acton, MA, USA) respectively, followed by colour reaction with 3′3′5′5′tetramethylbenzidine (TMB) and H2O2. Absorbance was read at 450–575 nm in an Emax microplate reader and antibody concentration was calculated by SOFTmax PRO software (Molecular Devices, Sunnyvale, CA, USA), according to standard curves included on each ELISA plate. IgG2 responses could not be detected for technical reasons.

BAFF levels were measured in baseline serum samples using BAFF quantikine ELISA (R&D Systems, Abingdon, UK), according to the manufacturer's instructions.

Statistical analysis

Data were analysed using GraphPadPrism version 5·0 (GraphPad software, San Diego, CA, USA). Mann–Whitney rank test, Wilcoxon's rank test and Spearman's rank test were performed for statistical analysis. A P-value <0·05 was considered statistically significant.

Results

IgG- and IgM influenza-specific antibody response measured by ELISA

Upon influenza vaccination, HC had a significant increase in IgM as well as IgG antibodies against both influenza strains (Fig. 1). RA patients treated with MTX also showed a good response to influenza for both isotypes.

Fig 1.

Fig 1

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Immunoglobulin (Ig)G and IgM anti-influenza response in healthy controls (HC), rheumatoid arthritis-methotrexate (RA-MTX) and RA-rituximab (RTX). Antibodies before (open symbols) and 28 days after (filled symbols) influenza vaccination were determined against H1N1 and H3N2 subunits by enzyme-linked immunosorbent assay (ELISA) in serum of 28 HC, 20 RA patients treated with MTX and 23 RA patients treated with RTX.

RA patients on RTX showed a low vaccination response compared to HC- and MTX-treated RA patients (Fig. 1). None of the responses in the RTX group were significant.

Correlation between HI and ELISA

To evaluate to what extent HI reflects ELISA results, we determined the correlation between HI and respective IgG and IgM ELISAs. IgG ELISA values correlated well with HI titres both in HC and in patient groups (Table 2). However, HI titres did not correlate with IgM levels in all samples (Table 2). There was no correlation in HC between H1N1-specific IgM antibody levels and H1N1-specific HI titres, whereas in RA-MTX the correlation between H3N2-specific IgM antibody levels and H3N2-specific HI titres was not significant, so the lack of correlation cannot be attributed to a specific influenza strain or to MTX or RTX therapy.

Table 2.

Correlations between HI titres and immunoglobulin (Ig)G and IgM enzyme-linked immunosorbent assay (ELISA) levels, respectively. Anti-influenza levels before and after vaccination were measured against H1N1 and H3N2 in HC (healthy controls), rheumatoid arthritis (RA) patients treated with methotrexate (RA-MTX) or rituximab (RA-RTX) and correlated with HI titres

IgG ELISA versus HI H1N1 H3N2
T = 0 T = 28 T = 0 T = 28
HC Spearman's r 0·85 0·50 0·51 0·51
P <0·0001 0·0091 0·0076 0·0083
RA-MTX Spearman's r 0·69 0·82 0·68 0·66
P 0·0007 <0·0001 0·0011 0·0017
RA-RTX Spearman's r 0·51 0·56 0·71 0·78
P 0·0129 0·005 0·0001 <0·0001
IgM ELISA versus HI H1N1 H3N2
T = 0 T = 28 T = 0 T = 28
HC Spearman's r 0·19 0·12 0·51 0·49
P n.s. n.s. 0·0081 0·0119
RA-MTX Spearman's r 0·71 0·5 0·32 0·32
P 0·0004 0·0236 n.s. n.s.
RA-RTX 0·6 0·54 0·51 0·62
P 0·0025 0·0081 0·013 0·0018
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n.s. = not significant.

To further investigate whether HI is determined mainly by IgG antibodies, sera of HC (n = 14) were depleted of IgM by incubation with agarose anti-IgM (cat. no. A9935; Sigma-Aldrich). After IgM depletion, IgG levels (original range 31–158 μg/ml) had not changed [percentage compared to untreated sample: average 103·2%, standard deviation (s.d.) 21·3%], while IgM levels (range untreated 3·7–224 μg/ml) were reduced (percentage compared to untreated sample: 14·8%, s.d. 9·9%). Performing HI with IgM depleted and untreated samples showed no changes in titre, confirming that HI is determined mainly by IgG antibodies.

IgG subclass response

Determination of the IgG subclass response by ELISA showed a significant IgG1 response in HC and both patient groups for H1N1 and H3N2 after vaccination, as can be seen in Table 3 (P < 0·001). Remarkably, the IgG1 response towards both influenza strains also reached significance in RA patients on RTX. H1N1-specific responses were seen in all groups for IgG3 (HC: P < 0·001, MTX: P < 0·01, RTX: P < 0·05). However, in contrast to both HC and RA-MTX patients, who had an increase in IgG3 response against H3N2 (HC: P < 0·01, MTX: P < 0·01), the RA-RTX group failed to reach an adequate increase in influenza-specific IgG3 after vaccination. IgG4 subclass response only showed a significant increase for H1N1 in HC (P = 0·02), and no increase in both patient groups.

Table 3.

Immunoglobulin (Ig)G1, IgG3 and IgG4 antibody levels to H1N1 and H3N2 before and 28 days after influenza vaccination in healthy controls (HC), rheumatoid arthritis (RA) patients treated with methotrexate (RA-MTX) or rituximab (RA-RTX)

Mean ± s.d. (μg/ml) Mean ± s.d. (μg/ml) P-value
HC T = 0 T = 28
IgG1 A/H1N1 50·58 ± 86·15 97·23 ± 96·67 <0·001
A/H3N2 42·81 ± 59·84 97·04 ± 122·40 <0·001
IgG3 A/H1N1 0·348 ± 0·899 1·169 ± 2·137 <0·001
A/H3N2 0·350 ± 0·852 0·948 ± 1·929 <0·001
IgG4 A/H1N1 0·195 ± 0·492 0·234 ± 0·610 0·022
A/H3N2 0·520 ± 1·573 0·578 ± 1·586 n.s.
RA-MTX
IgG1 A/H1N1 23·05 ± 17·99 83·00 ± 92·85 0·0001
A/H3N2 17·30 ± 14·53 98·25 ± 169·3 0·0001
IgG3 A/H1N1 0·134 ± 0·225 0·331 ± 0·440 0·0024
A/H3N2 0·152 ± 0·205 0·484 ± 0·625 0·0015
IgG4 A/H1N1 0·137 ± 0·217 0·221 ± 0·0396 n.s.
A/H3N2 0·197 ± 0·297 0·256 ± 0·484 n.s.
RA-RTX
IgG1 A/H1N1 25·87 ± 20·27 59·87 ± 78·32 0·0053
A/H3N2 25·39 ± 24·37 37·35 ± 43·90 0·0379
IgG3 A/H1N1 0·215 ± 0·617 0·303 ± 0·841 0·0467
A/H3N2 0·227 ± 0·561 0·296 ± 0·663 n.s.
IgG4 A/H1N1 0·079 ± 0·166 0·074 ± 0·150 n.s.
A/H3N2 0·095 ± 0·143 0·083 ± 0·164 n.s.
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s.d. = standard deviation; n.s. = not significant.

As mentioned previously, total IgG levels correlated well with HI titres. This seems due primarily to the IgG1 response, which forms the largest part of IgG. IgG1 levels correlated well with HI in the HC and patient groups for both influenza strains (P < 0·05). There was no correlation between HI levels and IgG3 anti-influenza levels in HC and RA-MTX patients, but HI titres correlated significantly with IgG3 anti-influenza levels in RA-RTX in both strains. No correlation was found between HI titres and IgG4 levels.

Early and late rituximab treatment groups

When patients within the RTX group were divided into patients who had received RTX 4–8 weeks before vaccination (early) and in those who had received RTX 6–10 months (late) prior to vaccination, an increase in influenza-specific IgG antibodies was observed in the ‘late’ RTX group only (Fig. 2). In the latter group, IgG to A/H1N1 increased from 48·9 ± 35·5 to 137·9 ± 127 (P = 0·002) and IgG to A/H3N2 increased from 39·6 ± 32·8 to 63·1 ± 49·8 (P = 0·001). The early group did not show a significant increase against either strain. In contrast, IgM response was not seen for either strain in both early and late groups (Fig. 2). In the late RTX group a significant increase for IgG1 and IgG3 was found for both H1N1 (respectively, P = 0·037 and P = 0·007) and H3N2 (respectively, P = 0·009 and P = 0·010). The early RTX group showed no increase in IgG1 or in IgG3 to either influenza strain.

Fig 2.

Fig 2

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Immunoglobulin (Ig)G (subclass) and IgM anti-influenza response in rheumatoid arthritis-rituximab (RTX) early and RA-RTX late. Antibodies before and 28 days after influenza vaccination were determined against H1N1 and H3N2 subunit by enzyme-linked immunosorbent assay (ELISA) in serum of 23 RA patients treated with RTX, divided into 11 RA-RTX early (RTX 4–8 weeks before vaccination) and 12 RA-RTX late (RTX 6–10 months before vaccination).

BAFF levels at baseline

As expected, we found high BAFF levels in RA patients who had been treated with RTX, both in early and late groups (Fig. 3a). BAFF levels in these patients were significantly increased compared to BAFF levels in HC and RA-MTX (all P < 0·001). The levels were [median (range)]: HC 0·66 ng/ml (0·14–1·04), RA-MTX 0·72 ng/ml (0·49–1·30, RA-RTXearly 2·56 ng/ml (1·28–4·58) and RA-RTXlate 2·18 ng/ml (1·28–4·83). There was no difference between BAFF levels in the early and late groups. A significant correlation was present between baseline BAFF levels and total IgG levels before (Fig. 3b) and after vaccination (P < 0·001 and P < 0·05, respectively), but not with IgA and IgM levels when the data of patients and controls were combined. In the separate (smaller) groups these correlations lost significance. Also, influenza-specific IgG levels after vaccination were correlated with BAFF levels in combined groups (P < 0·05), but not in separate groups. There was one exception: BAFF levels and IgG3-specific anti-influenza levels in RA-RTX patients were correlated negatively, P < 0·05, both for H1N1 and H3N2.

Fig 3.

Fig 3

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B cell-activating factor (BAFF) baseline levels in serum of healthy controls (HC), rheumatoid arthritis-methotrexate (RA-MTX) and RA-rituximab (RTX) (early, late). (a) Correlation between baseline BAFF levels and total IgG levels in HC, RA-MTX and RA-RTX (r = −0·33, P = 0·005). (b) BAFF levels (ng/ml) measured by enzyme-linked immunosorbent assay (ELISA) in serum of 28 HC, 20 RA patients treated with MTX and 23 RA patients treated with RTX, divided into 11 RA-RTX early (RTX 4–8 weeks before vaccination) and 12 RA-RTX late (RTX 6–10 months before vaccination).

Discussion

Seasonal influenza vaccination evokes a good response in healthy people and RA patients treated with MTX, as shown by a significant increase in influenza-specific IgG (IgG1 and IgG3) and IgM antibodies detected after vaccination. RA patients treated with RTX show a hampered response to influenza vaccination, especially for IgM antibodies to influenza.

Influenza vaccination is considered safe and efficacious (as determined by HI) in RA patients 21 even when treated with DMARDs and on anti-TNF therapy 11,12. Using ELISA we were able to unravel the humoral response after influenza vaccination. A significant increase in both IgG and IgM influenza-specific antibodies was found in HC and RA patients treated with MTX after vaccination. In line with previous results based on HI, no increase was seen in either IgG or IgM antibodies in RA-RTX patients. When patients were divided into early RTX and late RTX, a significant increase was seen in IgG antibodies but not in IgM antibodies in the late group. Previously it was shown that patients in the late group (those who received RTX 6–10 months before vaccination) had a modestly restored response measured by HI 12. Our results only show an increase in IgG antibodies. In these patients. Rehnberg et al. investigated vaccination responses to influenza vaccine and pneumococcal polysaccharides vaccine in RA patients 6 days before (n = 8) and 6 months after RTX treatment (n = 11) compared to RA patients on MTX treatment (n = 10) 22. They measured cellular response on day 6 and humoral response on day 21. Formation of influenza-specific B cells was lower in the post-RTX groups compared to the pre-RTX group and controls, and absence of influenza-specific IgG production was observed in 55% of the post-RTX group. These data corroborate ours, in that in our late RTX group, in which we included patients up to 10 months after RTX, we saw a modest restored IgG response. In a study by Pescovitz et al., the effect of RTX on human in-vivo antibody immune responses was investigated 23. The IgG and IgM response to a neoantigen was investigated as well as anti-tetanus, diphtheria, mumps, measles and rubella using ELISA. They showed that during the time of B lymphocyte depletion, RTX recipients had a decreased antibody response to neoantigens and significantly lower titres after recall immunization. With recovery of the B cells, immune responses returned to normal. They conclude that immunization during the time of B lymphocyte depletion, although ineffective, does not preclude a subsequent response to the antigen.

The IgG subclass ELISA demonstrated that the major part of the influenza-specific IgG response in all patient groups as well as HC consisted mainly of IgG1. In the HC IgG subclass response, after influenza vaccination with an inactivated subunit, vaccine has been compared to vaccination with a live attenuated vaccine 24. In young people both IgG1 and IgG3 responses could be demonstrated, but in older people (>58 years) there was only a significant IgG1 response 24. In another influenza vaccination study, only IgG1 and IgG2 antibodies were determined. A slight IgG2 response was seen in young children only after they had been ‘primed’ (had previous contact) 25. IgG2 responses remains controversial, because other studies failed to detect an increase both in young children and in elderly patients. In our study the average age of the patients was above 45 years, which could explain why the IgG4 response was lower, especially in patient groups that are considered to be immunocompromised because of disease and medication. The IgG4 response detected in HC might be explained by this influence of age, as the HC were younger than the patient groups. As stated before, the role of IgG4 is less clear from previous studies than IgG1 and IgG3, and the clinical consequences of the differences in IgG4 response remain to be elucidated.

BAFF levels were increased significantly in RTX-treated patients, both in the early and late groups. There was a significant correlation between BAFF levels and total IgG levels in combined HC and RA patients. After vaccination, only IgG3 influenza antibodies were correlated with BAFF levels in RTX-RA patients; no other correlations were seen between BAFF levels and response to influenza in patients and controls. This is in accordance with a recent study in which baseline BLys/BAFF levels were found not to correlate with humoral response to influenza vaccination in systemic lupus erythematosus (SLE) patients 26. Only patients with low BAFF (BLys) levels demonstrated an increased response, as we found in our study. BAFF is expressed by a variety of innate immune cells, such as dendritic cells, macrophages and neutrophils, whereas BAFF receptors are expressed mainly by B cells 27. Levels of BAFF appear to be critical for controlling peripheral B cell numbers and survival of autoreactive B cells so, in the case of low B cell numbers such as during RTX treatment, BAFF levels increase 27. This has been reported for RA patients whose BAFF levels increased after RTX infusion and remained elevated for at least 1–2 months 16. In primary Sjögren's syndrome patients treated with RTX it was shown that more transitional B cells were present in the reconstituted B cell population during the early recovery phase, corresponding to bone marrow-derived populations 28. This might explain why we see no correlation between BAFF levels and response to vaccination. Our study shows that influenza-specific IgG and IgM antibodies can be measured by ELISA, which has advantages over the HI method. Commercially available IgG and IgM anti-influenza type A or B ELISAs have been used in literature, but not compared to HI 29. Another study reported on the use of an IgG ELISA using the pandemic H1N1 HA protein as a coating antigen, and they found a concordance of 98·4% with HI 30. Recent studies show the advantages of ELISA methods over other methods as being quicker and easier to automate 31.

Our study has some limitations. As mentioned previously, our patient and HC groups are somewhat small, in particular when the RTX group is additionally divided into an ‘early’ and ‘late’ subgroup. Another limitation is the age difference in HC and patients. This might have influenced the IgG3 and IgG4 values, as has been found previously in other studies 24. Another possible confounder could be the non-standardized use of additional DMARDs. In the MTX group two patients used additional DMARDs; most of the RTX-treated patients were on MTX as well, and one was on corticosteroids. In the MTX group this does not seem to influence the response.

In conclusion, this study shows that the haemagglutination inhibition assay reflects primarily IgG influenza antibodies. It also shows that RA patients treated with RTX have a hampered IgG as well as IgM response after influenza vaccination. Although BAFF levels are increased significantly in RTX-treated patients, this does not have an effect on humoral response to vaccination. These results, in combination with data that show that RTX treatment does not (severely) interfere with cellular immunity and lack of increased infection rate in RTX-treated patients, strongly favour a central role of T cells in the defence against influenza virus.

Acknowledgments

The authors wish to thank Dr A. E. Voskuyl of University Medical Centre Amsterdam, Dr M. Blom of Radboud University Nijmegen Medical Centre and Dr A. P. Risselada of University Medical Centre Utrecht for providing patient materials. This work was supported by Roche Nederland and Solvay Pharmaceuticals. Both sponsors of the study had no role in study design, data collection, data analysis and writing of the manuscript. Submission was independent of the approval of the study by the sponsors.

Disclosure

The authors declare that they have no competing interests.

Author contributions

J. W., S. v. A., K. R. W., A. d. H. and M. B. contributed to the design of the study, interpretation of the data and drafting of the manuscript. S. v. A., K. R. W. and M. B. contributed to the inclusion of the patients. J. L. and G. H. performed experiments and contributed to the interpretation of the data. J. W. and M. B. wrote the manuscript. All authors read and approved the manuscript.

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