Evaluation of B-cell numbers in the study groups
At the time of immunization, the numbers of circulating B cells were evaluated by using flow cytometry. The numbers of B cells in percentages is calculated based on CD19+CD3- in the mononuclear population. The distribution in the post-RTX, pre-RTX, and controls is shown in Table .
Vaccine-specific cellular responses after RTX treatment
Cells secreting vaccine-specific Ig were enumerated 6 days after vaccination. At this time, the pre-RTX group had not received RTX and was identical to controls. Influenza = specific IgM B cells were prevalent in the pre-RTX group (median sfc/106 lymphocytes, 55[0 to 1704] and controls (sfc/106 lymphocytes 30[0 to 3,500]) as compared with the post-RTX group (sfc/106 lymphocytes, 3[0 to 277]). Significantly decreased levels of influenza-specific IgM-secreting B cells were detected in the post-RTX group versus controls (P = 0.046). In addition, when comparing the post-RTX group with the pre-RTX group, significantly lower levels were found (P = 0.044). No differences were found between the pre-RTX group and controls. The number of influenza-specific IgG- and IgA-producing cells was similar, irrespective of RTX treatment. Pneumo23-specific IgM-B cells were not found in any group. The number of B cells producing Pneumo23-specific switched Ig was lower (but not significant) as compared with the influenza-specific B cells. No other differences in Pneumo23-specific response were found between the groups.
Vaccine-specific humoral responses after RTX treatment
The levels of vaccine-specific Ig were compared on days 0 and 21 (Table ). We did not find any differences in the increased influenza-specific IgM levels between the groups. The levels of Pneumo23-specific IgM were elevated in controls compared with day 0 (P < 0.05). No significant increase of Pneumo23-specific IgM was found in the RTX-treated groups.
On day 21, the mean levels of influenza-specific IgG were significantly increased as compared with day 0 in the post-RTX group (Table ). When comparing influenza-specific IgG levels in the pre-RTX group and controls, no differences could be detected. A significant increase of Pneumo23-specific IgG was observed in pre-RTX group (P = 0.05) and controls (P = 0.037), whereas P23-specific IgG production in the post-RTX group was less pronounced. Pneumo23-specific IgG production was significantly higher in the pre-RTX group as compared with the post-RTX group (P = 0.027).
The post-RTX group was divided into patients treated with RTX once (n = 7) or repeatedly RTX treated (twice or more; n = 4), and the levels of vaccine-specific IgM and IgG were compared. We observed no differences in the levels of vaccine-specific Ig between the patients treated with RTX once as compared with those who were treated more than once, suggesting that repeated courses of RTX induced no further impairment of the vaccine-specific response.
Furthermore, dividing all RTX-treated patients into those previously treated with TNF-inhibitors (n = 15) and patients not treated with TNF-inhibitors (n = 4) revealed no differences between these groups, suggesting that previous treatment with TNF inhibitors does not affect our results.
IgG isotypes of vaccine-specific response to influenza and Pneumo23
The subclass analysis of IgG production showed that the control group produced IgG1 and IgG4 influenza-specific IgG (Figure ). In contrast, when combining the RTX-treated patients into one group, no subclass distinction in IgG production could be detected. However, a substantial part of these RTX-treated patients (five of 16; 31%) had no increase of any influenza-specific IgG subclass on day 21 after vaccination (Table ). Patients in the post-RTX group had the lowest proportion of responders to influenza of any IgG subclass (IgG1, four of 11; IgG2, six of 11; IgG3, six of 11; and IgG4, four of 11) (Table ). Interestingly, four patients in the post-RTX group did not respond in any IgG subclass against influenza immunization. In contrast, only one patient in the pre-RTX group and no one in the control group displayed a total lack of IgG-subclass increase (Table ).
Figure 1 Changes in IgG subclasses after protein and polysaccharide vaccination in rheumatoid arthritis (RA) patients before and after RTX treatment. (a) Changes at day 21 as compared with day 0 in immunoglobulin G (IgG)1-4 are shown after influenza vaccination. (more ...)
Individual responses of total IgG or IgG subclasses after influenza and pneumococcal polysaccharide immunization
For Pneumo23 vaccination, controls revealed a significant increase of IgG2 and IgG3, whereas post-RTX patients showed an isolated increase of IgG2, and pre-RTX patients, of IgG4 (Figure ). A total lack of IgG-subclass response against P23 was observed in four patients in the post-RTX group, but none in the pre-RTX group or in the control group (Table ).
A previous course of RTX did not affect the immune responses to influenza or P23, because only one of five patients that did not respond to influenza had a previous course of RTX, and two of four patients that did not respond to P23 had a repeated course of RTX (Table ). No difference in IgG subclasses was observed in patients with respect to previous treatment with TNF-α inhibitors.
Vaccine-specific production of Ig light chains after RTX treatment
We measured the development of vaccine-specific light-chains to evaluate functional B-cell populations in RTX-treated patients. On day 21, the levels of influenza-specific Ig containing λ-chains (Ig-λ) were elevated in all groups, irrespective of RTX treatment (Table ). In contrast, influenza-specific Ig-κ was elevated only in controls (P = 0.001), but neither in post-RTX nor in pre-RTX groups, suggesting that RTX may disrupt the production of Ig-κ. The pattern of Pneumo23-specific Ig-κ was similarly elevated in the pre-RTX group (P = 0.05) and in controls (P = 0.01).
The pattern of light-chain production correlated to vaccine-specific IgG levels. None of six post-RTX patients with the absence of an increase in influenza-specific IgG subclasses developed increase in influenza-specific Ig-| and Ig-| production. Only one of six patients had repeated RTX treatments; thus, no accumulation of light-chain suppression correlated with the number of RTX treatments.