Decreased influenza-specific B cell responses in rheumatoid arthritis patients treated with anti-tumor necrosis factor

Article (PDF Available)inArthritis research & therapy 13(6):R209 · December 2011with45 Reads
DOI: 10.1186/ar3542 · Source: PubMed
Abstract
As a group, rheumatoid arthritis (RA) patients exhibit increased risk of infection, and those treated with anti-tumor necrosis factor (TNF) therapy are at further risk. This increased susceptibility may result from a compromised humoral immune response. Therefore, we asked if short-term effector (d5-d10) and memory (1 month or later) B cell responses to antigen were compromised in RA patients treated with anti-TNF therapy. Peripheral blood samples were obtained from RA patients, including a subset treated with anti-TNF, and from healthy controls to examine influenza-specific responses following seasonal influenza vaccination. Serum antibody was measured by hemagglutination inhibition assay. The frequency of influenza vaccine-specific antibody secreting cells and memory B cells was measured by EliSpot. Plasmablast (CD19+IgD-CD27hiCD38hi) induction was measured by flow cytometry. Compared with healthy controls, RA patients treated with anti-TNF exhibited significantly decreased influenza-specific serum antibody and memory B cell responses throughout multiple years of the study. The short-term influenza-specific effector B cell response was also significantly decreased in RA patients treated with anti-TNF as compared with healthy controls, and correlated with decreased influenza-specific memory B cells and serum antibody present at one month following vaccination. RA patients treated with anti-TNF exhibit a compromised immune response to influenza vaccine, consisting of impaired effector and consequently memory B cell and antibody responses. The results suggest that the increased incidence and severity of infection observed in this patient population could be a consequence of diminished antigen-responsiveness. Therefore, this patient population would likely benefit from repeat vaccination and from vaccines with enhanced immunogenicity.

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RESEARC H ARTIC L E Open Access
Decreased influenza-specific B cell responses in
rheumatoid arthritis patients treated with anti-
tumor necrosis factor
James J Kobie
1
, Bo Zheng
1
, Peter Bryk
1
, Michael Barnes
2
, Christopher T Ritchlin
1
, Darren A Tabechian
1
,
Allen P Anandarajah
1
, R John Looney
1
, Ralf G Thiele
1
, Jennifer H Anolik
1
, Andreea Coca
1
, Chungwen Wei
1
,
Alexander F Rosenberg
1
, Changyong Feng
3
, John J Treanor
4
, F Eun-Hyung Lee
2
and Ignacio Sanz
1*
Abstract
Introduction: As a group, rheumatoid arthritis (RA) patients exhibit increased risk of infection, and those treated
with anti-tumor necrosis factor (TNF) therapy are at further risk. This increased susceptibility may result from a
compromised humoral immune response. Therefore, we asked if short-term effector (d5-d10) and memory (1
month or later) B cell responses to antigen were compromised in RA patients treated with anti-TNF therapy.
Methods: Peripheral blood samples were obtained from RA patients, including a subset treated with anti-TNF, and
from healthy controls to examine influenza-specific responses following seasonal influenza vaccination. Serum
antibody was measured by hemagglutination inhibition assay. The frequency of influenza vaccine-specific antibody
secreting cells and memory B cells was measured by EliSpot. Plasmablast (CD19+IgD-CD27hiCD38hi) induction was
measured by flow cytometry.
Results: Compared with healthy controls, RA patients treated with anti-TNF exhibited significantly decreased
influenza-specific serum antibody and memory B cell responses throughout multiple years of the study. The short-
term influenza-specific effector B cell response was also significantly decreased in RA patients treated with anti-TNF
as compared with healthy controls, and correlated with decreased influenza-specific memory B cells and serum
antibody present at one month following vaccination.
Conclusions: RA patients treated with anti-TNF exhibit a compromised immune response to influenza vaccine,
consisting of impaired effector and consequently memory B cell and antibody responses. The results suggest that
the increased incidence and severity of infection observed in this patient population could be a consequence of
diminished antigen-responsiveness. Therefore, this patient population would likely benefit from repeat vaccination
and from vaccines with enhanced immunogenicity.
Introduction
TNFais a potent pro-inflammatory cytokine produced
by macrophages, T, B, and dendritic cells, having pleio-
tropic effects on the immune system, including the
development and progression of autoimmune diseases.
TNF blockade has been extremely effective in treating
multiple inflammatory diseases, including rheumatoid
arthritis (RA); however, chronic blockade of TNF may
increase the risk of infections [1,2], including bacterial
pathogens such as tuberculosis, fungal infections, and
viral infections including herpes zoster and human
papillomavirus [2,3]. Furthermore, several studies have
reported reduced induction of serum antibodies in
patients treated with anti-TNF following vaccination
against influenza virus and pneumococcal bacteria [4-6].
Methotrexate (MTX), which inhibits folate metabolism
and promotes the production of immunosuppressive
extracellular adenosine, is commonly used to treat RA
[7]. Thus, anti-TNF treatment either alone or in combi-
nation with MTX may contribute to reduced immune
* Correspondence: ignacio_sanz@urmc.rochester.edu
1
Division of Allergy, Immunology and Rheumatology, University of Rochester
Medical Center, 601 Elmwood Avenue, Box 695, Rochester, NY, 14642, USA
Full list of author information is available at the end of the article
Kobie et al.Arthritis Research & Therapy 2011, 13:R209
http://arthritis-research.com/content/13/6/R209
© 2012 Kobie et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons
Attribution License (http://creative commons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, pro vided the original work is properly cited.
responses to infections and vaccination by limiting B
cell responses and subsequent development of protective
serum antibodies.
TNF impacts B-cell repertoire development and
homeostasis, as well as B cell responsiveness by multiple
direct and indirect mechanisms. This effect includes
direct modulation of B cell activation and survival
through nuclear factor (NF) B activation after the cyto-
kine binds surface TNFRI and TNFRII [8]. Surface-
bound TNF on activated macrophages and monocytes
can activate CD4+ T cells via TNFR and thus support
T-dependent B cell responses. Additionally, it has been
demonstrated that TNF mobilizes mouse bone marrow
B cells to the blood and spleen by suppressing stromal
CXCL12 retention signals in the bone marrow [9,10].
During an inflammatory response this could promote
bone marrow granulopoiesis and extramedullary lym-
phopoiesis, the former of which most likely plays a criti-
cal role in control of infection. Importantly, TNF has a
keyroleinfolliculardendritic cell organization and
function, and in germinal center reactions [11,12], and
we have previously demonstrated that TNF blockade
with etanercept in RA patients profoundly diminishes
the follicular dendritic cell network and disrupts germ-
inal center reactions [13]. Because germinal center reac-
tions are critical for optimal antibody induction, we
postulate that TNF blockade alters the effector and
memory B cell responses, contributing to increased risk
of infection and poor response to vaccination.
Following vaccination there is a transient effector B
cell response including the expansion of B cell plasma-
blasts, defined as CD19+IgD-CD27hiCD38hi cells that
can be readily observed in the peripheral blood and
strongly correlate with the vaccine-specific antibody-
secreting cell response [14-16]. In response to a recall
antigen such as influenza vaccine, the effector peak is
typically between five to seven days and slightly later in
a primary response, with a return to the steady state
within 14 days after immunization. This transient plas-
mablast population is highly enriched for B cells actively
secreting antibody against the immunogen [14,17] and
may contain precursors to the long-lived CD138+
mature plasma cells that reside in the bone marrow and
are the presumptive source of serum antibodies present
months and years following vaccination [15,18]. Addi-
tionally, following vaccination, antigen-specific memory
B cells develop, persist, and circulate throughout the
periphery, readied to differentiate into antibody-secret-
ing plasmablasts and plasma cells upon re-exposure to
antigen.
Anti-TNF has minimal effect on the ability of RA
patients to achieve the standard 40 or higher protective
titer after influenza vaccination [4,5,19,20]; however,
lower geometric mean titers (GMT) of antibody have
been observed [4-6]. These observations suggest that
treatment of RA patients with anti-TNF can result in
sub-optimal vaccine responses. In this study we specifi-
cally examined the impact of anti-TNF treatment of RA
patients on influenza-specific B cell responses. We
demonstrate that the majority of RA patients treated
with anti-TNF have a decreased influenza-specific effec-
tor B cell response that correlates with diminished
development of memory B cells and serum antibodies.
Materials and methods
Patients
We enrolled 261 subjects, including 164 patients with
diagnosed RA and 97 healthy control subjects at the
University of Rochester Medical Center from 2006 to
2010 who were all receiving seasonal inactivated triva-
lent influenza vaccine (TIV) as standard-of-care. Patients
provided signed written informed consent. All proce-
dures and methods were approved by the Research Sub-
jects Review Board at the University of Rochester
Medical Center.
Sampling
Peripheral blood was obtained from subjects at one time
point prior to receiving TIV, and then at one and six
months post-vaccination. This cycle was repeated for two
seasons for each subject. Based on subject willingness,
availability, and logistical constraints, a subset of subjects
provided two additional samples collected following
2009-2010 TIV immunization; one obtained on day five
to day seven post-vaccination, and another obtained day
eight to day ten post-vaccination. A limited number of
samples were also obtained from subjects day five to day
seven following 2010-2011 TIV immunization.
PBMC were isolated within two hours of sampling
using CPT tubes (Becton Dickinson, Franklin Lakes, NJ,
USA). Tubes were immediately inverted 8 to 10 times
and processed according to manufacturersinstructions.
Peripheral blood mononuclear cells (PBMCs) were cryo-
preserved and stored in liquid nitrogen. Serum was col-
lected, aliquotted and stored at -80°C. All sample
processing was performed in a blinded manner.
Hemagglutination inhibition assay
A standard hemagglutination inhibition assay was per-
formed in a blinded manner as previously described
[21]. Briefly, serial two-fold serum dilutions were
assayed against influenza H1N1, H3N2, and B using
strains contained in corresponding years TIV vaccine or
appropriately matched closely related strain.
Memory B cell culture
Total B cells were isolated from fresh PBMC by negative
magnetic bead selection of non-B cells according to
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manufacturers protocol (Miltenyi Biotec, Auburn, CA,
USA) and cryopreserved. B cell samples were thawed,
washed once in culture media (RPMI containing 10%
heat-inactivated fetal bovine serum and antibiotic/anti-
mycotic), and 30,000 cells added per well of a 96-well
round-bottom plate in 200 μl culture media containing
2.5 μg/ml CpG ODN 2006 (Oligos Etc, Wilsonville, OR,
USA) and 10 ng/ml IL-2 (Peprotech, Rocky Hill, NJ,
USA). Cells were cultured for four days at 37°C and 5%
CO2.
Antibody-secreting cell Elispots
The frequency of influenza antigen-specific antibody-
secreting cells (ASCs) was measured by Elispot as pre-
viously described [16,22]. Briefly, 96-well Elispot plates
(MAIPS4510 96 well, Millipore, Bedford, MA, USA)
were coated overnight at 4°C in a humidified chamber
with either: TIV Influenza Virus Vaccine (6 μg/mL,
Sanofi Pasteur Inc., Swiftwater, PA, USA), anti-human
IgG (5 μg/mL, Jackson Immunoresearch, West Grove,
PA, USA), or anti-human IgM (5 μg/mL, Jackson
Immunoresearch, West Grove, PA, USA). These anti-
gens and capture antibodies were diluted in sterile PBS
to above concentrations. Bovine serum albumin (2%) in
sterile PBS was used as an irrelevant antigen. Coated
plates were blocked with RPMI and 8% fetal bovine
serum for two hours and incubated at 37°C for 18 to 20
hours with 300,000, 100,000, and 33,333 PBMC or with
25,000 and 2,500 cultured B cells per well in triplicate.
For total-IgG and IgM plates, 100,000, 33,000, and
11,000 PBMC or 2,500 and 250 cultured B cells per well
were added. After incubation, cells were aspirated and
plates were washed with PBS with 0.1% Tween (PBST).
Bound antibodies were detected with alkaline phospha-
tase-conjugated anti-human IgG or anti-human IgM
antibody (1 μg/mL, Jackson Immunoresearch) for two
hours and developed with VECTOR Blue, Alkaline
Phosphatase Substrate Kit III (Vector Laboratories, Bur-
lingame, CA, USA). Spots in each well were counted in
a blinded manner using the CTL immunospot reader
(Cellular Technologies Ltd, Shaker Heights, OH, USA).
Flow cytometry
Cryopreserved peripheral blood lymphocytes were
thawed and stained with anti-CD19-APC-Cy7 (SJ25C1,
BD Biosciences, San Diego, CA, USA) anti-CD20-Alex-
aFluor 700 (2H7, Biolegend, San Diego, CA, USA),
anti-CD3-PerCP-Cy5.5 (SP34-2, BD, anti-IgD-FITC
(IA6-2, BDanti-IgM-PE-Cy5 (G20-127anti-CD27-
Qdot605 (CLB-27/1, Invitrogen, Grand Island. NY,
USA), anti-CD38-PE-Cy7 (HIT2, ebioscience, San
Diego, CA, USA), anti-CD24-PE-AlexaFluor 610 (SN3,
Invitrogenanti-CD1c-PE (AD5-8E7, Miltenyi Biotec,
Auburn, CA, USA), anti-CD45R/B220-APC (RA3-6B2,
BD), and Live/Dead fixable violet dead cell stain (Invi-
trogen). One-to-two million events per sample were
collected on an LSRII instrument (BD Biosciences) and
analysis performed in a blinded manner using FlowJo
software (Treestar, Inc, Ashland, OR, USA). Total
PBMC were gated on lymphocytes using FSC and SSC.
To exclude dead cells, and T cells, Live/Dead stain,
and anti-CD3 were used, respectively. Plasmablasts
were identified within the CD19
+
B cell population as
IgD-CD24-CD27++CD38++. Fresh lymphocytes were
also stained in the same manner from samples
obtained days five to seven after immunization with
2010-2011 TIV.
Statistical analysis
Fishers exact test was used to compare differences in
the induction of serum antibodies and TIV-specific
memory B cells among groups from multiple years. For
multiple-year composite data, an individual subject may
have provided data for two years. Kruskal-Wallis test
was used to compare plasmablast, ASC, and serum anti-
body responses to the 2009-2010 TIV between groups.
For correlative analysis of 2009-2010 HAI average
change for each subject was determined as: ((H1 1mo/
H1 base) + (H3 1mo/H3base) + (B 1mo/B base))/3.
Spearman one-tailed correlation co-efficient was used to
measure the correlation of two variables. Statistical ana-
lyses were performed using Prism 5.0 software (Graph-
Pad Software, La Jolla, CA, USA) and SAS 9.2 (SAS
Institute Inc., Cary, NC, USA).
Results
Study design and patient demographics
This multi-year study was designed to broadly assess the
impact of anti-TNF therapy on B cell responses to influ-
enza. Between 2006 and 2010 peripheral blood was
obtained prior to (0), one, and six months following vac-
cination with seasonal trivalent inactivated influenza
vaccine (TIV) administered as standard-of-care. For
most subjects, this cycle was repeated for two seasons.
AsshowninTable1,theRA+aTNFgroup(RA+aTNF)
was comprised of patients receiving adalimumab
(Humira), etanercept (Enbrel), or infliximab (Remicade)
also as standard-of-care. Many of these subjects were
concomitantly treated with MTX. The RA+MTX group
consisted of patients treated with MTX, but not anti-
TNF or any other biological therapy. There was no sta-
tistical difference in the MTX dose between the RA
+aTNF and RA+MTX dose (not shown). The RA group
(RA) consisted of patients not receiving MTX, anti-
TNF, or any other biological therapy, and was predomi-
nantly comprised of recently diagnosed patients. A
diverse group of healthy controls (HC) was enrolled
encompassing a broad range of ages.
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Influenza-specific antibody and memory B cell response
To determine the serum antibody response to influenza
vaccination the hemagglutination inhibition assay was
performed. RA patients treated with anti-TNF had lower
GMT of serum antibody compared with healthy controls
in all study years (Figure 1). Specifically, at one month
following vaccination, RA patients treated with anti-
TNF had on average throughout all the study years 50%,
65%, and 30% lower H1, H3, and B GMT, respectively,
compared with HC (Figure 1). At six months following
vaccination, RA patients treated with anti-TNF had on
average throughout all the study years 46%, 46%, and
31% lower H1, H3, and B GMT, respectively, compared
with HC (Figure 1). Although lower GMT were
observed in the RA+MTX group compared with the HC
group, it was less consistent than that observed in the
RA+aTNF group. Although variability throughout the
study years was also observed in the post-vaccination
GMT of the untreated RA group, interestingly, however,
increased GMT were observed at several timepoints
compared with the HC group. Notably, most subjects
had serum titers that reached sero-protective levels at
one month following vaccination as defined by HAI titer
of 40 or higher [see Additional data file 1].
We next determined the response rate to the influenza
vaccine as defined by the frequency of subjects within
each group that developed a four-fold or greater
increase in HAI titer at one month or six months post
vaccination over baseline. This analysis mitigated the
impact of baseline GMT variation, which is likely to be
due to differences in past influenza vaccination and
natural infection among individuals, and facilitated
group comparisons across multiple study years. A
greater proportion of the untreated RA group responded
toH1andH3ascomparedwiththeHCgroupatboth
one and six months post-vaccination (Figure 2a), and
consistent with the annual results discussed above, this
increase was sustained after six months in the untreated
RA group. By contrast, fewer of the RA+aTNF patients
responded to H1 at one and six months compared with
the HC and RA groups. This difference only reached
significance when the RA+anti-TNF group was com-
pared with the untreated RA group. The RA+aTNF
group and the RA+MTX group both had a lower pro-
portion of patients responding to H3 compared with
HC and RA. Both reached significance compared with
RA, but only the RA+aTNF group was significantly
lower than HC. Both the RA+MTX and RA+aTNF
groups had a significantly lower proportion of patients
that responded to influenza B compared with the HC
and untreated RA groups. Therefore, the serum anti-
body response against multiple components of seasonal
influenza vaccine is impaired in RA patients treated
with anti-TNF. This effect may result from altered
development, maintenance, or quality of influenza-speci-
fic plasma cells. As the influenza vaccine response is lar-
gely a secondary immune response, TNF blockade may
be compromising the memory B cell response, perhaps
by altering their maintenance or responsiveness.
To determine if anti-TNF therapy similarly affects the
influenza-specific memory B cell response, TIV IgG Eli-
Spots were performed from CpG and IL-2 stimulated B
Table 1 Cohort demographics
RA+aTNF RA+MTX RA Healthy Controls
N61 70 33 97
Age, mean +/- SD years 55.4 +/- 12.3 58.4 +/-12.2 57.1 +/-13.8 39.8 +/- 13.6
% female 82% 77% 64% 63%
Disease duration < 1 year 5% 17% 45% NA
Disease duration > 3 years 93% 60% 47% NA
Adalimumab n (%) 9 (15) NA NA NA
Infliximab n (%) 17 (28) NA NA NA
Etanercept n (%) 35 (57) NA NA NA
MTX n (%) 49 (80) 70 (100) 0 (0) NA
MTX, dose +/- SD mg/wk 15.2 +/- 4.3 16.5 +/- 4.0 NA NA
corticosteroid n (%) 18 (29.5) 26 (37.1) 10 (30.3) NA
corticosteroid, dose +/- SD mg/d 5.5 +/- 3.5 4.9 +/- 2.0 10.3 +/-6.1 NA
ESR mean (range) 21 (4-70) 22 (2-83) 20 (2-74) NA
HAQ mean (range) 0.71 (0.00-2.22) 0.73 (0.00-2.75) 0.46 (0.00-1.63) NA
VAS mean (range) cm 2.8 (0.0-9.8) 3.3 (0.0-7.5) 2.8 (0.0-7.1) NA
Morning stiffness mean (range) min 81 (0-780) 104 (0-1440) 59 (0-180) NA
Race non Caucasian n (%) 8 (13) 6 (9) 2 (6) 12 (12)
ESR, erythrocyte sedimentation rate; HAQ, health assessment questionnaire; MTX, methotrexate; NA, not applicable; SD, standard deviation; VAS, visual analogue
score.
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cells from a subset of patient samples. At baseline
approximately 2.4% and 1.3% of the total IgG memory B
cells for the HC and RA+aTNF groups respectively were
TIV specific, but this difference was not significant (P=
0.097) [see Additional data file 2]. When examined as
response following TIV, similar to the serum antibody
response, the frequency of RA patients treated with
anti-TNF that had a four-fold or greater increase in
TIV-specific memory B cells at one month post-vaccina-
tion was significantly lower than both the HC and RA
groups. Moreover, the influenza-specific memory B cell
frequencies continued to decrease disproportionally at
six months in anti-TNF treated patients as compared
with all other groups. A modest increase was also
observed in the frequency of untreated RA with a four-
fold or greater increase in TIV-specific IgG memory B
cells compared with the HC group (40% vs. 35% at one
month, 33% vs. 31% at six months; Figure 2b). No sig-
nificant difference was observed between the RA+MTX
group and HC and RA groups. These results indicate
that TNF blockade adversely impacts influenza-specific
memory B cell responses to vaccination in RA patients
both at the induction and maintenance phases of the
memory response.
Effector response to influenza vaccine
In HC, we have previously observed that the peak fre-
quency of influenza-specific ASCs in the peripheral
blood present shortly following vaccination correlates
with the subsequent increase in serum hemagglutination
inhibition (HAI) antibodies [16]. It is suggested that
ASC are enriched in the peripheral blood CD19+IgD-
CD27++CD38++ plasmablast population. We therefore
asked if this effector response to influenza vaccination is
also impacted by anti-TNF. In addition to baseline and
one month samples, two peripheral blood samples were
obtained from a subset of RA patients and healthy con-
trols; one sample between day five and day seven, and a
second between day eight and day ten following admin-
istration of the 2009-2010 seasonal influenza vaccine.
Untreated RA patients had a significantly greater change
in the frequency of plasmablasts compared with healthy
controls (Figures 3a and 3b). By contrast, RA patients trea-
ted with anti-TNF had a significantly decreased induction
of plasmablasts at days five to seven compared with both
HC and untreated RA patients (Figure 3b). This effect was
observed in both fresh and frozen samples and also con-
firmed in a limited analysis of samples following 2010-
2011 TIV (Figure 3a and data not shown). To precisely
Figure 1 Serum hemagglutination-inhibition antibody titers. Peripheral blood was obtained prior to (baseline), and one and six months
following vaccination with trivalent influenza vaccine (TIV) throughout multiple years. Influenza-specific serum antibody was measured by
hemagglutination inhibition assay and geometric mean titer (GMT) determined for the indicated influenza subtype. GMT (upper 95% confidence
interval (CI)/lower 95% CI) indicated. Relative difference in GMT among rheumatoid arthritis (RA) groups as compared with the healthy control
(HC) group at same timepoint indicated by color. Cells with bold lines indicate significant difference (P< 0.05) compared with HC group at
same timepoint. N, number of subjects measured per year. MTX, methotrexate.
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examine the induction of TIV-specific IgG and IgM ASC
after vaccination PBMC were assayed by EliSpot. Signifi-
cantly less TIV-specific IgG ASC were observed at days
five to seven in RA patients treated with anti-TNF as com-
pared with healthy controls (Figure 3c). Furthermore RA
patients treated with anti-TNF had significantly decreased
TIV IgG ASC at both days five to seven and days eight to
ten as compared with untreated RA patients. Similar,
although non-significant trends were observed for TIV-
specific IgM ASC (data not shown). These results indicate
the effector B cell response to influenza vaccine is
impaired in RA patients treated with anti-TNF. Decreased
plasmablast and ASC responses were observed in the RA
+MTX group; however, likely due to limited sample num-
ber, significance was only reached when comparing days
five to seven plasmablast change compared with untreated
RA patients.
Examination of the early serum antibody response
demonstrated that as soon as days eight to ten, RA
patients treated with anti-TNF exhibited significantly
lower HAI titers against H1, H3, and B compared with
healthy controls, and significantly decreased titers to H1
and B as compared with untreated RA patients (Figure
4). At one month post-vaccination RA patients treated
with anti-TNF continued to have significantly decreased
HAI titers to H3 and B compared with healthy controls,
and decreased HAI titers to H1, and B compared with
untreated RA patients. Differences in the HAI titer of
the RA+MTX as compared with HC or untreated RA
group were not consistent. These results suggest RA
patients treated with anti-TNF have a substantially
impaired effector B cell and antibody response.
Correlation between effector and memory responses
against influenza vaccine
We previously determined that the peak of TIV-specific
peripheral blood ASC shortly after vaccination correlates
with the peak in HAI titer after vaccination [16].
Figure 2 Induction of serum antibodies and memory B cells.(a) The frequency of subjects within each group that had a four-fold or greater
increase in serum hemagglutination inhibition assay (HAI) titer over baseline was determined in 2006/2007 through 2009/2010 influenza seasons, and
combined results indicated. (b) Total B cells were isolated, cultured with CpG and IL-2 for four days, and trivalent influenza vaccine (TIV) and total IgG
specific EliSpots performed to determine the frequency of TIV-specific memory B cells. The frequency of subjects that had a four-fold or greater
increase in the frequency of TIV-specific IgG memory B cells over baseline is presented. The red star indicates significant difference (P<0.05)compared
with healthy control (HC) group, the blue star indicates significant difference as compared with rheumatoid arthritis (RA) group. An individual subject
may have provided data from multiple study years. N, the cumulative number of paired subject events measured throughout the multiple years.
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Figure 3 Plasmablast and antibody secreting cell response. The frequency of peripheral blood plasmablasts (IgD-CD24-CD27++CD38++)
among total CD19+ B cells was determined by flow cytometry at baseline, day five to day seven, and day eight to day ten following
vaccination with seasonal trivalent influenza vaccine (TIV). (a) Identification of plasmablasts from representative subjects at day five to day seven
following 2010-2011 TIV. Plots are gated on live, CD3-CD19+IgD-CD24- cells. (b) Fold change in plasmablast frequency over baseline following
2009-2010 TIV is indicated. (c) TIV IgG-specific EliSpots were performed on total peripheral blood mononuclear cells (PBMC) following 2009-2010
TIV. The red star indicates significant difference (P< 0.05) as compared with healthy control (HC) group, the blue star indicates significant
difference as compared with rheumatoid arthritis (RA) group. Each symbol represents an individual study subject, red line indicates group mean.
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However, it is not clear if the effector response mea-
sured by EliSpot for ASC or by flow cytometry for plas-
mablasts correlates with the frequency of TIV-specific
memory cells and how this may be impacted in RA
patients treated with anti-TNF. We thus asked if the
decreased effector response to influenza vaccine in RA
patients treated with anti-TNF is associated with the
observed decrease in memory B cell and serum antibody
on a per-patient basis. We observed a significant posi-
tive correlation of the IgG TIV-specific ASC measured
at days five to seven post-vaccination with the frequency
of IgG TIV-specific memory B cells measured at one
month (r = 0.64, P= 0.0003; Figure 5a) and as expected
with the change in HAI titer at one month (r = 0.74, P
< 0.0001; Figure 5b). Additionally, the induction of
CD19+IgD-CD27++CD38++ plasmablasts at days five to
seven also significantly correlated with the frequency of
IgG TIV-specific memory B cells (r = 0.67, P= 0.0006;
Figure 5c) and the change in HAI titer at one month (r
= 0.42, P= 0.0396; Figure 5d).
The subjects that had the lowest effector and memory
B cell and antibody responses were primarily RA
patients treated with MTX and/or anti-TNF, suggesting
that these therapies directly impact the patientsacute
responsiveness to TIV and subsequent generation of
TIV-specific memory B cells and serum antibodies.
Discussion
We have demonstrated that RA patients treated with
anti-TNF have impaired B cell and antibody responses
to seasonal influenza vaccine. This suboptimal response
includes lower serum HAI antibodies, which is consis-
tent with previous studies [4-6] and also diminished
induction of influenza-specific effector and memory B
cell responses, which have not been previously
recognized.
The impact of anti-TNF therapy was most evident
during the effector phase (day 5 to day 10) than the
memory phase (one to six months) of the B cell
response. This difference most likely reflects the sensi-
tive resolution of vaccine response provided at these
early time-points. Whereas resolution of the influenza-
specific B cell and antibody responses at one month or
later after vaccination is confounded by variation in
amounts of pre-existing memory B cells and antibody
present among individuals, potentially underestimating
the responsiveness to a single vaccination event. The
strong positive correlation observed between the effector
B cell response from day five to day seven after vaccina-
tion and the memory B cell and antibody response at
one month suggests a predictive value for examining the
effector B cell response as an early biomarker for the
development of memory B cells and antibody. The
reduced effector and memory B cell response we
observed in anti-TNF-treated patients, may contribute
to the increased risk and severity of infection observed
in anti-TNF-treated patients.
Treatment with anti-TNF is likely to target multiple
aspects of the vaccine response that are contributing to
the decreased B cell response observed. Previously, we
demonstrated that lymphoid architecture is altered in
RA patients treated with etanercept, including the
destruction of the FDC network and disruption of germ-
inal centers and corresponding decrease in peripheral
blood memory B cells [13]. This dysregulation may be
preventing influenza-specific B cells from participating
in optimal germinal center reactions, thereby compro-
mising the magnitude and quality of the effector and
memory influenza-specific B cell response. Furthermore,
as autocrine TNF enhances B cell proliferation [23], and
TNF is produced in abundance by plasma cells [24];
TNF blockade may inhibit this process, contributing to
Figure 4 Influenza-specific serum antibodies following 2009-2010 TIV. Peripheral blood was obtained prior to (0) and at multiple time
points within one month following vaccination with 2009 to 2010 seasonal trivalent influenza vaccine (TIV). Influenza-specific serum antibody
was measured by hemagglutination inhibition assay (HAI) and geometric mean titer (GMT) determined. The red star indicates significant
difference (P< 0.05) compared with healthy control (HC) group at same timepoint, the blue star indicates significant difference compared with
the rheumatoid arthritis (RA) group at same timepoint. N, number of subjects measured for each group.
Kobie et al.Arthritis Research & Therapy 2011, 13:R209
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Page 8 of 12
the reduced development of influenza-specific memory
B cells and plasmablasts that we observed. A possible
additional and not mutually exclusive point of impact
for TNF blockade is limiting the maintenance of influ-
enza-specific plasma cells, because TNF enhances
plasma cell survival in vitro [25], and has been suggested
to be important for plasma cell survival in vivo [26,27].
It would be of particular interest to determine if TNF
blockade is merely blocking the activation and
expansion of influenza-specific plasmablasts and mem-
ory B cells or promoting the development of a distinct
short-lived effector population that does not produce
antibody, nor can develop adequately into antibody-pro-
ducing cells.
Diminished B cell and antibody responses may also
result from impaired monocyte, dendritic, and T cell
function by TNF-blockade that has been reported, limit-
ing the supportive, yet potentially critical roles these
Figure 5 Comparison of effector and memory response following 2009-2010 TIV.(a) Trivalent influenza vaccine (TIV) IgG antibody-
secreting cells (ASC) at day five to day seven vs. frequency of IgG TIV-specific memory B cells and (b) change in hemagglutination inhibition
(HAI) titer as measured at one month. Fold change in IgD-CD27++CD38++ plasmablasts at (c) day five to day seven vs. frequency of IgG TIV-
specific memory B cells and (d) change in HAI titer as measured at one month. Each symbol represents an individual study subject. Healthy
controls (HC; n= 9 to 11), rheumatoid arthritis (RA; n= 3 to 5), RA+methotrexate (MTX; n= 3 to 4), RA+aTNF (n= 3 to 6). Spearman one-tailed
correlation co-efficient (r) and significance (P) is indicated.
Kobie et al.Arthritis Research & Therapy 2011, 13:R209
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cells play in the induction of a robust B cell response.
The known suppressive effect of anti-TNF on memory
and effector T cell functions such as IFNgand IL-2 pro-
duction [28], and increased Treg function [29,30] may
negatively impact the B cell response. A limitation of
the current study is the inability to adequately assess the
various anti-TNF agents individually for their impact on
influenza vaccine response. We expect that slight differ-
ences in the B cell response to vaccination may become
apparent in a large multi-anti-TNF agent comparison
study; however, it is uncertain that it would be feasible
to dissect differences that occur as a result of mode of
action, from those that could result from the different
routes of administration or dosing schedules for the var-
ious agents.
Interestingly, we observed subtle indications of
enhanced B cell responsiveness to the influenza vaccine
in RA patients not treated with MTX or anti-TNF as
compared with the healthy control subjects. This out-
come may reflect an inflammatory environment that
could be present in these patients, resulting from the
disease process, contributing in an adjuvant-like manner,
thereby enhancing the germinal center reaction, and
consequently the plasmablast and memory B cell
responses. Additionally, rheumatoid factor-expressing B
cells exhibit an enhanced ability to internalize antigen-
immunoglobulin complexes and subsequently present
the antigen to specific T cells [31], raising this possibi-
lity as a potential mechanism of enhanced vaccine
responses in some RA patients. Future studies with
greater numbers of untreated RA patients are warranted
to adequately substantiate and dissect these questions
further. Such studies may elucidate additional mechanis-
tic aspects of the inflammatory processes in RA disease
and provide strategies to enhance vaccine responsiveness
in the general population.
Although a decreased response to the influenza vac-
cine was observed in RA patients treated with anti-
TNF, it is important to emphasize that vaccination did
increase serum antibody and presumed protection
from infection. Furthermore, recent European League
Against Rheumatism (EULAR) recommendations indi-
cate that influenza vaccination should be strongly con-
sidered, and that vaccination can be administered
during the use of anti-TNF therapy [32]. The anti-
TNF treated patient population may benefit from the
high-dose influenza vaccine recently licensed for indi-
viduals aged 65 years and older, and warrants further
investigation. Moreover, our results indicate that every
effort should be made to provide annual influenza
immunization and indicated vaccination updates to
RA patients before anti-TNF therapy is initiated. It is
also possible that the efficacy of annual influenza vac-
cination in anti-TNF treated patients could be
improved by timing immunization with the decline in
biological effect of the corresponding agent. This
question could be addressed by studying the relative
efficacy of immunization provided just before the next
dose of anti-TNF. The clinical value of examining
anti-TNF and vaccination timing is substantiated by a
recent study by Elkayam at al. which demonstrated
that RA patients treated with infliximab three weeks
prior to influenza vaccination had a reduced serum
antibody response as compared with those patients
treated with infliximab on the day of vaccination [33].
In patients with particularly high risk (i.e. those
patients with previous history of severe infection, low
baseline HAI titers, and/or other co-morbidities) it
maybereasonabletodelaythenextdoseofanti-TNF
drug to provide a longer window of developing a pro-
tective response.
Conclusions
RA patients treated with anti-TNF have a diminished B
cell and antibody response to the influenza vaccine. This
impairment is evident in the suppressed plasmablast
response, and limited induction of memory B cells and
serum antibody, highlighting the role of TNF in this
process. The implications of this finding should be con-
sideredinthecontextoftheincreasedsusceptibilityto
infections observed in anti-TNF treated patients and
suggest the need for vaccines with enhanced immuno-
genicity for these patient populations.
Additional material
Additional file 1: Additional Table 1. HAI seroprotection proportion.
Peripheral blood was obtained prior to (baseline), and one and six
months following vaccination with trivalent influenza vaccine (TIV)
throughout multiple years. Influenza-specific serum antibody was
measured by hemagglutination inhibition assay (HAI) for the indicated
influenza type. The number of subjects with HAI titer of 40 or higher/
total number of subjects (%) are indicated. Relative difference in
geometric mean titer (GMT) among rheumatoid arthritis (RA) groups as
compared with the healthy control (HC) group at same time point
indicated by color. Cells with bold outline indicate significant difference
(P< 0.05) as compare with the HC group at same timepoint.
Additional file 2: Additional Figure 1. TIV-specific IgG memory B
cell frequency. Total B cells were isolated at baseline and one and six
months following immunization with 2006/2007 through 2009/2010
trivalent influenza vaccine (TIV), cultured with CpG and IL-2 for four days,
and TIV and total IgG specific EliSpots performed to determine the
frequency of TIV specific memory B cells. An individual subject may have
provided data from multiple study years. No statistical difference (P<
0.05) was detected among groups at individual time points.
Abbreviations
ASC: antibody-secreting cells; GMT: geometric mean titer; HAI:
hemagglutination inhibition assay; HC: healthy controls; MTX: methotrexate;
NF: nuclear factor; PBMC: peripheral blood mononuclear cells; PBS:
phosphate buffered saline; RA: rheumatoid arthritis; TIV: trivalent influenza
vaccine; TNF: tumor necrosis factor.
Kobie et al.Arthritis Research & Therapy 2011, 13:R209
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Page 10 of 12
Acknowledgements
We extend our sincere gratitude to Sally Quataert and the staff of the
Rochester Human Immunology Center for sample processing, Theresa
Fitzgerald for performing the hemagglutination inhibition assays, Denise
Kaminski for critical review of the manuscript, and for the effort and
commitment to the study demonstrated by the clinical coordinators and
study participants. This work was supported by the NIH/NIAID Rochester
Center for the Biodefense of Immunocompromised Populations
HHSN2662005500029C (N01-AI50029).
Author details
1
Division of Allergy, Immunology and Rheumatology, University of Rochester
Medical Center, 601 Elmwood Avenue, Box 695, Rochester, NY, 14642, USA.
2
Division of Pulmonary & Critical Care Medicine, University of Rochester
Medical Center, 601 Elmwood Avenue, Box 692, Rochester, NY, 14642, USA.
3
Department of Biostatistics and Computational Biology, University of
Rochester Medical Center, 601 Elmwood Avenue, Box 630, Rochester, NY,
14642, USA.
4
Division of Infectious Disease, University of Rochester Medical
Center, 601 Elmwood Avenue, Box 689, Rochester, NY, 14642, USA.
Authorscontributions
All authors were involved in drafting the article or revising it critically for
important intellectual content, and all authors read and approved the final
version to be published. JJK 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. The study was conceived and designed by JJK and IS. JJK, BZ,
MB, PB, APA, DAT, RJL, RGT, JHA, AC, CTR, and JJT contributed to the
acquisition of data. Data was analyzed and interpreted by JJK, AFR, JJT, CF,
FE-HL and CW and IS.
Competing interests
JJK has received grant/research support from Biogen. FE-HL has received
grant/research support from Trellis Biosciences, Inc. JT has received grant/
research support from Protein Sciences Corp., GlaxoSmithKline, Sanofi, Pfizer,
Bavarian Nordic, PaxVax, Ligocyte, and Vaxinnate and has received honoraria
from ITS, Inc. and Novartis. IS has received grant/research support from
Biogen and has performed consulting work for Genetech and
GlaxoSmithKline.
Received: 8 June 2011 Revised: 21 October 2011
Accepted: 16 December 2011 Published: 16 December 2011
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Cite this article as: Kobie et al.: Decreased influenza-specific B cell
responses in rheumatoid arthritis patients treated with anti-tumor
necrosis factor. Arthritis Research & Therapy 2011 13:R209.
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    • "Studies of inactivated trivalent influenza vaccines have shown that although TNFi treatment moderately decreases humoral responses, the patients still develop protective antibody levels [7,8,[17][18][19], and the response can even equal that in healthy controls [16]. The aforementioned studies were conducted in RA patients, yet one study also included patients with other chronic illnesses [8]. "
    [Show abstract] [Hide abstract] ABSTRACT: Background: Tick-borne Encephalitis (TBE) is endemic in south-eastern Sweden as well as in the Baltic regions, Central Europe and Russia. Ageing and immunosuppressed individuals are more prone to severe disease and neurological complications. We assessed the immunogenicity of TBE-vaccine in rheumatoid arthritis (RA) patients treated with tumor necrosis factor-inhibitors (TNFi) and/or methotrexate (MTX). Methods: TBE vaccine, FSME-Immune(®) or Encepur(®), was administered to non-immune RA patients as well as age and gender matched healthy controls. Individuals <60 years of age were given three doses at month 0, 1, 12. Individuals ≥60 years old were given an additional priming dose at month 3, i.e. a total of four doses. Tick-borne encephalitis neutralizing antibodies were assessed by a rapid fluorescent focus inhibition test. Results: The study population consisted of 66 patients and 56 age and gender matched healthy controls. Median age was 58.5 years. The patients were either treated with TNFi (n=16), TNFi+MTX (n=36) or MTX (n=14). After the last TBE-vaccine dose, given one year after the first, 39% of the patients compared to 79% of the healthy controls had seroprotective levels (p=<0.05). Conclusions: Standard TBE-vaccine schedule does not confer enough immunogenicity in this group of immunosuppressed patients, who should be carefully informed about a higher risk for vaccination failure and risk of infection when exposed in high-endemic areas.
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    • "Samples from normal donors were obtained at the URMC. Peripheral blood mononuclear cells, plasma, and sera were isolated and cryopreserved as previously described [20]. "
    [Show abstract] [Hide abstract] ABSTRACT: Potent HIV-1 specific broadly neutralizing antibodies (BNA) are uncommon in HIV infected individuals, and have proven hard to elicit by vaccination. Several, isolated monoclonal BNA are polyreactive and also recognize self-antigens, suggesting a breach of immune tolerance in persons living with HIV (PLWH). Persons with systemic lupus erythematosus (SLE) often have elevated levels of autoreactive antibodies encoded by the VH4-34 heavy chain immunoglobulin gene whose protein product can be detected by the 9G4 rat monoclonal antibody. We have recently found that levels of these "9G4+" antibodies are also elevated in PLWH. However, the putative autoreactive nature of these antibodies and the relationship of such reactivities with HIV neutralization have not been investigated. We therefore examined the autoreactivity and HIV neutralization potential of 9G4+ antibodies from PLWH. Results show that 9G4+ antibodies from PLWH bound to recombinant HIV-1 envelope (Env) and neutralized viral infectivity in vitro, whereas 9G4+ antibodies from persons with SLE did not bind to Env and failed to neutralize viral infectivity. In addition, while 9G4+ antibodies from PLWH retained the canonical anti-i reactivity that mediates B cell binding, they did not display other autoreactivities common to SLE 9G4+ antibodies, such as binding to cardiolipin and DNA and had much lower reactivity with apoptotic cells. Taken together, these data indicate that the autoreactivity of 9G4+ antibodies from PLWH is distinct from that of SLE patients, and therefore, their expansion is not due to a general breakdown of B cell tolerance but is instead determined in a more disease-specific manner by self-antigens that become immunogenic in the context of, and possibly due to HIV infection. Further studies of 9G4+ B cells may shed light on the regulation of B cell tolerance and interface between the generation of specific autoreactivities and the induction of antiviral immunity in persons living with HIV.
    Full-text · Article · Dec 2013
    • "The involvement of the influenza pathway in RA, however, has not been reported before and may provide new clues to understand the pathophysiology mechanism of the disease. Indeed, a recent study showed that RA patients have an increased risk of infection although the increased susceptibility to infections could not be attributed to a compromised humoral immune response [25]. "
    [Show abstract] [Hide abstract] ABSTRACT: Genome-wide association studies (GWAS) led to the identification of numerous novel loci for a number of complex diseases. Pathway-based approaches using genotypic data provide tangible leads which cannot be identified by single marker approaches as implemented in GWAS. The available pathway analysis approaches mainly differ in the employed databases and in the applied statistics for determining the significance of the associated disease markers. So far, pathway-based approaches using GWAS data failed to consider the overlapping of genes among different pathways or the influence of protein–interactions. We performed a multistage integrative pathway (MIP) analysis on three common diseases - Crohn's disease (CD), rheumatoid arthritis (RA) and type 1 diabetes (T1D) - incorporating genotypic, pathway, protein- and domain-interaction data to identify novel associations between these diseases and pathways. Additionally, we assessed the sensitivity of our method by studying the influence of the most significant SNPs on the pathway analysis by removing those and comparing the corresponding pathway analysis results. Apart from confirming many previously published associations between pathways and RA, CD and T1D, our MIP approach was able to identify three new associations between disease phenotypes and pathways. This includes a relation between the influenza-A pathway and RA, as well as a relation between T1D and the phagosome and toxoplasmosis pathways. These results provide new leads to understand the molecular underpinnings of these diseases. The developed software herein used is available at http://www.cogsys.cs.uni-tuebingen.de/software/GWASPathwayIdentifier/index.htm.
    Full-text · Article · Oct 2013
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