Host Differences in Influenza-Specific CD4 T Cell and B
Cell Responses Are Modulated by Viral Strain and Route
Aarthi Sundararajan1., Lifang Huan2., Katherine A. Richards1, Glendie Marcelin3, Shabnam Alam1,
HyeMee Joo4, Hongmei Yang5, Richard J. Webby3, David J. Topham1, Andrea J. Sant1, Mark Y. Sangster1*
1David H. Smith Center for Vaccine Biology and Immunology, Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, New
York, United States of America, 2Department of Microbiology, University of Tennessee, Knoxville, Tennessee, United States of America, 3Department of Infectious
Diseases, Division of Virology, St. Jude Children’s Research Hospital, Memphis, Tennessee, United States of America, 4Baylor Institute for Immunology Research, Baylor
University Medical Center, Dallas, Texas, United States of America, 5Department of Biostatistics and Computational Biology, University of Rochester Medical Center,
Rochester, New York, United States of America
The antibody response to influenza infection is largely dependent on CD4 T cell help for B cells. Cognate signals and
secreted factors provided by CD4 T cells drive B cell activation and regulate antibody isotype switching for optimal antiviral
activity. Recently, we analyzed HLA-DR1 transgenic (DR1) mice and C57BL/10 (B10) mice after infection with influenza virus
A/New Caledonia/20/99 (NC) and defined epitopes recognized by virus-specific CD4 T cells. Using this information in the
current study, we demonstrate that the pattern of secretion of IL-2, IFN-c, and IL-4 by CD4 T cells activated by NC infection is
largely independent of epitope specificity and the magnitude of the epitope-specific response. Interestingly, however, the
characteristics of the virus-specific CD4 T cell and the B cell response to NC infection differed in DR1 and B10 mice. The
response in B10 mice featured predominantly IFN-c-secreting CD4 T cells and strong IgG2b/IgG2c production. In contrast, in
DR1 mice most CD4 T cells secreted IL-2 and IgG production was IgG1-biased. Infection of DR1 mice with influenza PR8
generated a response that was comparable to that in B10 mice, with predominantly IFN-c-secreting CD4 T cells and greater
numbers of IgG2c than IgG1 antibody-secreting cells. The response to intramuscular vaccination with inactivated NC was
similar in DR1 and B10 mice; the majority of CD4 T cells secreted IL-2 and most IgG antibody-secreting cells produced IgG2b
or IgG2c. Our findings identify inherent host influences on characteristics of the virus-specific CD4 T cell and B cell responses
that are restricted to the lung environment. Furthermore, we show that these host influences are substantially modulated
by the type of infecting virus via the early induction of innate factors. Our findings emphasize the importance of
immunization strategy for demonstrating inherent host differences in CD4 T cell and B cell responses.
Citation: Sundararajan A, Huan L, Richards KA, Marcelin G, Alam S, et al. (2012) Host Differences in Influenza-Specific CD4 T Cell and B Cell Responses Are
Modulated by Viral Strain and Route of Immunization. PLoS ONE 7(3): e34377. doi:10.1371/journal.pone.0034377
Editor: Steven M. Varga, University of Iowa, United States of America
Received January 13, 2012; Accepted March 1, 2012; Published March 23, 2012
Copyright: ? 2012 Sundararajan et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by a grant (HHSN266200700008C) from the National Institutes of Health/National Institute of Allergy and Infectious Diseases
(http://www.niaid.nih.gov). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
. These authors contributed equally to this work.
Studies of mouse models of influenza A virus infection have
produced a comprehensive but as yet incomplete picture of
disease pathogenesis and the innate and adaptive antiviral
mechanisms that contribute to viral clearance and recovery.
The initial phase of influenza virus replication in epithelial cells,
local macrophages, and dendritic cells triggers the rapid release
of a range of cytokines and chemokines with antiviral and
pro-inflammatory activity [1,2]. In addition to limiting viral
replication in the respiratory tract, these processes are critical
for the optimal activation of antigen-specific B and T cells and
the development of adaptive immunity . The ultimate
elimination of infectious virus from the respiratory tract is
dependent on B and T cells through mechanisms such as the
destruction of virus-infected cells by infiltrating cytotoxic CD8 T
cells and the antiviral activity of progressively increasing antibody
(Ab) levels .
Optimal virus-specific Ab production by B cells following
influenza infection is dependent on CD4 T cell help. Although
some antiviral Abs can be generated in the absence of CD4 T cells,
Ab production is substantially more vigorous and effective
following collaborative interactions between CD4 T cells and B
cells [5,6]. CD4 T cells provide cognate signals and secreted
factors that drive B cell activation and differentiation and regulate
Ab isotype switching. After cognate interactions of peptide:MHC
class II (MHC II)-bearing B cells with CD4 T cells, activated B
cells may differentiate via the extrafollicular pathway to rapidly
generate a population of short-lived virus-specific Ab-secreting
cells (ASCs), or they may enter B cell follicles and initiate germinal
center (GC) reactions where long-lasting populations of ASCs and
memory B cells expressing high affinity antiviral Abs are formed
PLoS ONE | www.plosone.org1 March 2012 | Volume 7 | Issue 3 | e34377
. The progression of B cells through the GC reaction is
dependent on a second phase of a cognate T cell help delivered by
T follicular helper (Tfh) cells . The CD4 T cell response to
influenza infection has long been regarded as ‘‘Th1-polarized’’
and characterized by high levels of IL-2 and interferon (IFN)-c
secretion [9,10]. A Th1-type cytokine profile fits well with the
typical influenza-specific B cell response, which includes a
predominance of the IgG2a (IgG2c in some mouse strains) and
IgG2b isotypes. IFN-c promotes the expression of IgG2a/IgG2c
and IgG2b by B cells [11,12].
Recently, we used HLA-DR1 transgenic (DR1) mice to define
HLA-DR1-restricted epitopes recognized by influenza virus-
specific CD4 T cells [13,14]. DR1 mice were infected with the
H1N1 influenza virus A/New Caledonia/20/99 (NC) and
multiple strong epitopes were identified in the 5 viral proteins
analyzed, HA, NA, NP, M1, and NS1. IL-2 production and
cytokine ELISpot assays were used for identification of specific
CD4 T cells. The current study was initiated to relate the
specificity and frequency of CD4 T cells induced by NC infection
in DR1 mice with the secretion of IFN-c and IL-4, as well as IL-2.
Surprisingly, the pattern of secreted cytokines was inconsistent
with the expected Th1-polarized response and included a
relatively high proportion of IL-4-secretors among the specific
CD4 T cells. This prompted us to examine a conventional mouse
strain with a defined NC-specific CD4 T cell repertoire and to
conduct a parallel analysis of the influenza-specific B cell response.
Our findings indicate that host factors modulate influenza-specific
CD4 T cell and B cell responses in a lung-restricted fashion. In
addition, we show that these lung-specific differences can be
overridden by the type of infecting virus via early effects on the
Materials and Methods
Experiments involving animals were performed in accordance
with the recommendations in the Guide for the Care and Use of
Laboratory Animals of the National Institutes of Health and with
the approval of Animal Care and Use Committees at the University
of Rochester (protocol numbers 2006-029, 2006-030, and 2008-
023) and the University of Tennessee (protocol number 1283).
Infectious stocks of the following viruses were grown and titrated
as previously described: (i) human influenza virus A/New Caledo-
nia/20/99 (H1N1) , (ii) influenza A/Puerto Rico/8/34 (H1N1)
(PR8) and A/HK/X31 (H3N2) (X31) , and (iii) murine
gammaherpesvirus 68 (MHV68) . Purified viruses for use in
immunoassays were prepared by differential centrifugation and
sucrose banding of virus stocks. Purified PR8 and X31 purchased
from Charles River (Wilmington, MA) were used in some
experiments.InfluenzaNCfor useasanimmunogenwas inactivated
by b-propiolactone treatment of stock virus prior to purification and
exposure to UV light after purification. Complete inactivation was
confirmed by absence of cytopathic effect in Vero cell monolayers
inoculated with the treated virus. Viral protein concentrations were
determined using the Bio-Rad protein assay (Bio-Rad, Hercules,
CA). All virus preparations were stored at 280uC.
HLA-DR1 transgenic mice (B10.M/J-TgN-DR1)  were
obtained from D. Zaller (Merck) through Taconic laboratories and
C57BL/10J (B10) mice were purchased from The Jackson
Laboratory (Bar Harbor, ME). Mice were maintained under
specific pathogen-free conditions and were used at 8–16 wk of age.
Male and female mice were used for the analysis of CD4 T cell
responses following influenza infection and for the measurement of
influenza titers in the lung; otherwise, female mice were used in all
Immunizations and Sampling
Mice were anesthetized with Avertin (2,2,2-tribromoethanol)
given intraperitoneally before all immunizations. Infectious virus
for intranasal (i.n.) inoculation was diluted in Dulbecco’s PBS and
a 30 ml volume was applied to the external nares. For intramuscular
(i.m.) immunization, a total dose of 20 mg of inactivated virus was
given in two injections, each of 10 mg (inoculum volume of 50 ml in
PBS), into the tibialis anterior muscle of each leg. A plastic sleeve
over the needle controlled the depth of injection.
Anesthetized mice were exsanguinated via the retro-orbital plexus
before tissue sampling. Lymph nodes and spleen were collected and
gently disrupted between the frosted ends of microscope slides to
generate single-cell suspensions. Red blood cells were removed from
spleen preparations by ammonium chloride lysis. Lungs to be
titrated for infectious virus were homogenized in 1 ml HBSS
containing antibiotics and 0.1% BSA. Homogenates were clarified
bycentrifugation,and supernatants were stored at280uC.Lungsfor
the measurement of tissue levels of cytokines and chemokines were
perfused with ice-cold PBS before removal and were processed as
previously described . Briefly, lungs were homogenized in T-
PER Tissue Protein Extraction Reagent (Pierce, Rockford, IL)
containing Complete Mini Protease Inhibitor Cocktail tablets
(Roche, Indianapolis, IN). Homogenates were centrifuged and the
supernatants were stored at 280uC.
Flow Cytometry for B Cell Phenotyping
Cell suspensions were stained with the following directly
conjugated reagents at previously determined optimal concentra-
tions: anti-CD4/CD8 PE-Cy7 (RM4-5/53-6.7), anti-CD45R/
B220 PerCP (RA3-6B2), anti-CD95/Fas Alexa 647 (Jo2), and anti-
CD138 allophycocyanin (281-2), (BD Biosciences), anti-CD19 PE-
Cy5.5 (1D3) (eBioscience), and PNA FITC (Vector Laboratories).
Live/dead fixable violet staining kit (Invitrogen) was used to
discriminate dead cells. Data were acquired using an LSR II flow
cytometer (BD Biosciences) and analyzed using FlowJo software
(TreeStar). ASCs were defined as CD42CD82CD19+B220int
CD138+cells ; GC B cells were defined as CD42CD82
ELISpot Assay for Cytokine-Secreting Cells
Cytokine production by CD4 T cells was analyzed by ELISpot
assay as previously described [13,21]. Briefly, 96-well filter plates
were coated with 2 mg/ml purified rat anti-mouse IL-2, IFN-c, or
IL-4 (clones JES6-1A12, AN18, and 11B11 respectively; BD
Biosciences), then washed and blocked. CD4 T cells enriched by
negative selection from the pooled tissues of 3–5 mice were plated
at a range of concentrations (typically 50,000–300,000 cells/well),
together with DAP-3 fibroblasts expressing the HLA-DR1 MHC
class II protein (35,000 cells/well) or with T cell-depleted syngenic
splenocytes (500,000 cells/well). Selected peptides or peptide pools
were included in the cultures (10 mM of each peptide/well) and
plates were incubated for 18–20 hr at 37uC with 5% CO2. Spots
representing cytokine-secreting cells were visualized by developing
the plates, in sequence, with biotinylated rat anti-mouse cytokine
Abs (clones JES6-5H4 (IL-2), XMG1.2 (IFN-c) and BVD6-24G2
(IL-4); BD Biosciences), alkaline phosphatase-conjugated strepta-
vidin, and alkaline phosphatase substrate. A previously defined
immunodominant peptide (HA-75) was included in most ELISpot
Modulation of T and B cell Responses to Influenza
PLoS ONE | www.plosone.org2 March 2012 | Volume 7 | Issue 3 | e34377
assays to control for the degree of CD4 T cell priming. Peptides
used in the ELISpot assay were obtained from the NIH/NIAID
Biodefense and Emerging Infections Research Repository and
were handled as previously described .
ELISpot Assay for Ab-Secreting Cells
Virus-specific ASCs were enumerated by ELISpot assay as
previously described . Briefly, plates were coated with purified
virus and single-cell suspensions were plated and incubated.
Alkaline phosphatase-conjugated goat anti-mouse Abs with
specificity for immunoglobulin (Ig) isotypes (Southern Biotechnol-
ogy, Birmingham, AL) were used in combination with the
substrate 5-bromo-4-chloro-3-indolyl phosphate to generate spots.
Virus-specific Ab levels in sera were determined by ELISA as
previously described . Briefly, serial 3-fold sample dilutions
were added to virus-coated plates, and bound Ab was detected
with alkaline phosphatase-conjugated goat anti-mouse Abs with
specificity for Ig isotypes or IgG (Southern Biotechnology) and p-
nitrophenyl phosphate substrate. The virus-specific serum Ab titer
is expressed as the reciprocal of the highest dilution giving an
absorbance value more than twice that for simultaneously titrated
samples from naı ¨ve mice.
Total serum levels of IgM, IgG1, IgG2b, IgG2c, IgG3, and IgA
were determined by a sandwich ELISA . Concentrations were
calculated from curves constructed using purified mouse Ig
standards (Southern Biotechnology).
Viral titers in lung homogenates were determined by 50% tissue
culture infective dose assay using Madin Darby canine kidney cells
CD4 T Cell Depletion
Mice were depleted of CD4 T cells by intraperitoneal
administration of 200 mg of the CD4 T cell-specific mAb GK1.5
twice before and twice after infection. Non-depleted control mice
were given an isotype-matched mAb.
Multiplex and ELISA for Lung Cytokines and Chemokines
Lung homogenate supernatants were assayed for a panel of
cytokines and chemokines using a 32-plex multiplex kit (Millipore)
according to the manufacturer’s instructions. The concentration of
IFN-a was measured using a commercially available ELISA kit
(PBL, Piscataway, NJ).
Cytokine and chemokine concentrations in lung homogenates
were analyzed by two-way ANOVA after log transformation to
stabilize variance, followed by the Tukey-Kramer correction to
control type I error. Other comparisons of group means were
performed using a two-tailed Student’s t-test or nonparametric
Mann–Whitney U-test for unpaired samples. Statistically signifi-
cant P values are indicated by one (P,0.05), two (P,0.01), or
three (P,0.001) asterisks.
Contrasting patterns of CD4 T cell cytokine production in
DR1 and B10 mice following NC infection
In our analysis of the specificity of CD4 T cells after influenza
NC infection in DR1 mice, we identified a broad CD4 T cell
repertoire, with recognition of multiple HLA-DR1-restricted
epitopes in each of the HA, NA, NP, M1, and NS1 viral proteins.
This information enabled us to now ask whether the expected
Th1-type profile of secreted cytokines after influenza infection was
influenced by T cell epitope specificity. CD4 T cells purified from
the mediastinal lymph node (MedLN) and spleen on day 10 after
NC infection of DR1 mice were tested for their ability to secrete
IL-2, IFN-c, or IL-4 after stimulation with defined peptide
epitopes. The patterns of secretion of these cytokines by peptide-
reactive CD4 T cells were similar in the MedLN and spleen and
largely independent of peptide specificity and the magnitude of the
peptide-specific response (Fig. 1, A and B). Generally, within each
peptide-specific CD4 T cell population a slightly larger proportion
of the cells produced IL-2 and similar proportions produced IFN-c
The comparable proportions of IL-4- and IFN-c-secretors
among the virus-specific CD4 T cells in DR1 mice was surprising
and inconsistent with the Th1-polarized response expected after
influenza infection. We therefore conducted a similar analysis in
B10 mice, a conventional mouse strain in which we had analyzed
the response to NC infection and defined CD4 T cell-recognized
epitopes in same set of viral proteins analyzed in DR1 mice .
B10 mice share the non-MHC genetic background of DR1 mice.
As was the case in DR1 mice, the pattern of cytokine production
by virus-specific CD4 T cells in B10 mice was independent of
epitope specificity and response magnitude (Fig. 1, C and D).
However, in contrast to the result in DR1 mice, IFN-c-secreting
cells clearly predominated over IL-2-secreting cells in B10 mice
and IL-4-secreting cells formed a relatively small proportion. The
contrasting patterns of cytokine secretion in DR1 and B10 mice
are summarized in Fig. 1, E and F, which show the proportions of
IL-2-, IFN-c-, and IL-4-secretors among combined peptide-
specific CD4 T cells.
The B cell response to NC infection in DR1 mice is
delayed and IgG1-biased
The contrasting patterns of cytokine production by virus-
specific CD4 T cells in DR1 and B10 mice after NC infection
prompted us to investigate B cell response differences, since
cytokines produced by T cells during cognate interactions with B
cells play a key role in directing the expression of particular Ig
isotypes. In both DR1 and B10 mice, NC infection was sublethal
and infectious virus was eliminated from the lung after a clear
phase of replication. Virus titers in the lung on days 1, 3, 5, and 8
after infection were similar in DR1 and B10 mice, indicating
comparable antigen loads (Fig. 2). By day 8, a strong virus-specific
IgM and IgG ASC response had developed in the draining
MedLN of B10 mice, but IgM ASCs predominated in the MedLN
of DR1 mice (Fig. 3, A, B, C, D). The response in the MedLN of
B10 mice was typical of previously described influenza-specific B
cell responses in several mouse strains and included a strong
IgG2b and IgG2c component. When a NC-specific IgG response
developed in DR1 mice, it was strongly biased to IgG1 production
(see MedLN and spleen on day 14). The percentage of CD19+B
cells in the MedLN on day 8 was similar in DR1 and B10 mice.
There was a trend towards a higher proportion of CD19+cells
with the B220intCD138+phenotype of ASCs in B10 mice, but the
difference from DR1 mice was not significant (Fig. 3E and Fig.
S1). The proportion of GC cells (PNA+Fas+) in the B220+cell
population was significantly higher in DR1 mice (Fig. 3F and Fig.
S1), suggesting stronger GC activity. Infection with a higher dose
of NC did not substantially modify the differences between DR1
and B10 mice in the features of the NC-specific ASC response in
the MedLN (Fig. 3, G and H). We concluded that the different
Modulation of T and B cell Responses to Influenza
PLoS ONE | www.plosone.org3 March 2012 | Volume 7 | Issue 3 | e34377
patterns of Ig isotype expression in DR1 and B10 mice after NC
infection fit closely with the contrasting patterns of cytokine
production by virus-specific CD4 T cells, since IL-4 and IFN-c act
reciprocally in promoting expression of IgG1 and IgG2b/IgG2c,
ELISA measurements of NC-specific serum Abs were consistent
with the pattern of ASC generation in lymphoid tissues (Fig. 4).
Early virus-specific IgG levels were significantly higher in B10
compared with DR1 mice, primarily reflecting increased IgG2b,
IgG2c, and IgG3 production. IgM levels did not follow this pattern
and were significantly higher in DR1 mice on days 14 and 21. NC-
specific serum Ig levels were significantly higher in B10 compared
with DR1 mice on day 8 and similar at later sampling times.
The quality of the NC-specific B cell response in DR1 mice
depends on the form of immunization
Total serum levels of IgM and the IgG isotypes were similar in
uninfected DR1 and B10 mice (Fig. S2), suggesting that the
prominence of IgG1 in the response of DR1 mice to NC infection
did not reflect an intrinsic bias impacting B cell responses in
general. We therefore compared B cell and CD4 T cell responses
in DR1 and B10 mice following i.m. administration of purified
Figure 1. The CD4 T cell response to NC infection. (A–D) Cytokine production by peptide-specific CD4 T cells. DR1 (A and B) and B10 (C and D)
mice were infected intranasally with 40,000 EID50NC. Enriched CD4 T cells from the MedLN and spleen were analyzed on day 10 after infection.
Frequencies of CD4 T cells secreting IL-2, IFN-c, or IL-4 were determined by ELISpot assay after in vitro stimulation with antigen-presenting cells and
individual 17-mer peptides. Peptide designations (x-axis) include the viral proteins of origin (HA, NA, NP, M1, and NS1). Results are normalized to spot
counts per 106CD4 T cells and are shown as the mean+SEM for 2–6 independent experiments for each peptide. Cells from at least three mice were
pooled for each experiment. (E, F) Proportions of peptide-specific CD4 T cells secreting IL-2, IFN-c, or IL-4. Results are compiled from the data shown
in A–D and represent 4 (MedLN) or 6 (spleen) independent experiments evaluating 3–12 (MedLN) or 12–20 (spleen) individual peptides. The
mean+SEM is shown.
Modulation of T and B cell Responses to Influenza
PLoS ONE | www.plosone.org4 March 2012 | Volume 7 | Issue 3 | e34377
inactivated NC virus particles, a strategy that allowed us to
compare CD4 T cell responses to the same epitopes after a
different mode of immunization. Virus-specific IgG responses were
largely comparable in DR1 and B10 mice; the responses followed
similar kinetics and were characterized by a predominance of the
IgG2b/IgG2c isotypes (Fig. 5, A, B, C, D). Not only was there no
IgG1 bias in the response in DR1 mice, the IgG2c response in the
IliLN was generally more vigorous in DR1 than in B10 mice.
In both DR1 and B10 mice after i.m. immunization, the highest
proportion of peptide-specific CD4 T cells secreted IL-2, with
smaller proportions secreting IFN-c- or IL-4 (Fig. 5, E, F, G, H).
Our findings point to the lung environment as a key element in the
delay and IgG1 bias of the IgG response to NC infection in DR1
mice. Furthermore, they indicate that a predominance of IFN-c-
secreting cells is not a prerequisite for an IgG2b/IgG2c-biased B
The virus-specific B cell and CD4 T cell response to lung
infection in DR1 mice is modulated by the infecting virus
To determine whether the response to NC infection in DR1 mice
was independent of virus type, we compared B cell responses in
DR1 and B10 mice after infection with other influenza viruses (PR8
and X31), and with a non-influenza virus (MHV68) that also
replicates in the lung. In contrast to what was observed after NC
infection, infection of DR1 mice with PR8, X31, or MHV68
generated a virus-specific B cell response in the MedLN that was
largely comparable to the response in B10 mice (Fig. 6, A, B, C, D,
Figure 2. Replication of NC in the lung. DR1 and B10 mice were
sampled at intervals after intranasal infection. Virus titers are
represented as log10TCID50/0.2 ml of lung homogenate. The mean 6
SE is shown for 3–4 (days 1, 3, 5, and 14) and 10–20 (days 8 and 10)
individual mice per group. * P,0.05.
Figure 3. The B cell response to NC infection. (A–D) Virus-specific ASC frequencies. DR1 (A and B) and B10 (C and D) mice were infected
intranasally with 40,000 EID50NC. Virus-specific ASC frequencies in the MedLN and spleen were determined by ELISpot assay at intervals after
infection. (E, F) Flow cytometric analysis of ASCs and germinal center B cells. MedLN cells were analyzed on day 8 after intranasal NC infection (40,000
EID50dose) of DR1 and B10 mice. ASC frequencies (E) represent the proportion of B220intCD138+cells after gating on live CD42CD82CD19+cells.
Germinal center B cell frequencies (F) represent the proportion of PNA+Fas+cells among live CD42CD82B220+cells. Representative staining profiles
are shown in figure S1. (G, H) Virus-specific ASC frequencies in the MedLN of DR1 and B10 mice after intranasal infection with a high dose (100,000
EID50) of NC. Fig. 3 data sets depict the mean+SE for 3–8 individual mice per group. ** P,0.01.
Modulation of T and B cell Responses to Influenza
PLoS ONE | www.plosone.org5 March 2012 | Volume 7 | Issue 3 | e34377
E, F). Most notably, the IgG response of DR1 mice was not biased
to IgG1 production. Interestingly, the early IgM ASC response was
stronger in DR1 compared with B10 mice after PR8, X31, and
MHV68 infection, a trend that was also evident after NC infection.
The response to PR8 infection in DR1 and B10 mice was
compared in greater detail. Virus titers in the lungs were similar on
days 6 and 8, indicating similar viral loads (Fig. 6G). Flow
cytometric analysis of MedLN cells on day 8 showed similar
percentages of CD19+B cells and similar proportions of CD19+
cells that were B220intCD138+(Fig. 6H), indicating comparable
early ASC generation. The proportion of GC cells (PNA+Fas+) in
the B220+cell population was significantly higher in DR1
compared with B10 mice (Fig. 6I), as was also the case after NC
infection, suggesting an inherent tendency towards a stronger GC
response in DR1 mice regardless of the infecting virus. Early
serum levels of PR8-specific IgG were significantly lower in DR1
than in B10 mice (Fig. 6J), consistent with the situation after NC
infection. Thus, modulation of the IgG isotype expression profile
in DR1 mice by PR8 infection was not associated with an
increased rate of IgG production. Although the high frequency of
IgM ASCs in DR1 mice after PR8 infection raised the possibility
of an unusually strong T-independent Ab response to this virus
, we found that the formation of both IgM and IgG ASCs
specific for PR8 in the MedLN of DR1 mice on day 8 was largely
eliminated by CD4 T cell depletion (Fig. S3), indicating a
dependence on CD4 T cell help.
We next asked how the quality of the B cell response after PR8
infection related to the pattern of cytokines produced by virus-
specific CD4 T cells. Purified CD4 T cells from the MedLN on
day 10 after PR8 infection were tested for their ability to produce
cytokines after stimulation with peptides known to be strong
epitopes in NC and conserved between NC and PR8. In contrast
to the response to NC infection, IFN-c-secreting cells were
consistently more frequent than IL-2-secreting cells in DR1 mice
after PR8 infection (Fig. 7, A and C). The profile in B10 mice after
PR8 infection was similar to that after NC infection, with clearly
the majority of virus-specific CD4 T cells secreting IFN-c (Fig. 7, B
and D). Our findings demonstrate that the type of infecting virus
modulates the virus-specific B cell response to infection of the
respiratory tract in DR1 mice, perhaps via an increase in the
proportion of IFN-c-secreting CD4 T cells.
Antigen-independent effects of lung infection modulate
the virus-specific B cell response in DR1 mice
We hypothesized that the contrasting B cell responses in DR1
mice following infection with NC compared with PR8 and the
other viruses tested reflected differences in the early induction of
innate factors that set the character of the immune response. To
test this idea, we asked whether the response to NC infection
would be modified by concurrent MHV68 infection. MHV68 was
selected for this experiment because it is not cross-reactive with
influenza virus at the B cell level  and it elicits a B cell response
in DR1 mice with a clear IgG2c predominance among switched
Ab isotypes (Fig. 6C). Cohorts of DR1 and B10 mice were infected
with NC plus MHV68, or with NC only, and the B cell response
on days 8 and 10 was assessed by ELISpot assay using plates
coated with NC or MHV68 (Fig. 8, A, B, C, D). The NC-specific
response in DR1 mice infected with NC alone was modified by
concurrent MHV68 infection and resembled the response in B10
mice infected with NC alone. Notably, the NC-specific IgG
response in co-infected DR1 mice was not delayed relative to the
response in B10 mice and the predominant IgG isotype was
IgG2c, not IgG1. Serum levels of NC-specific IgG and Ig on day 8
were also consistent with a more rapid NC-specific response in co-
infected DR1 mice compared with those infected with NC alone
(Fig. 8, E and F). This experiment demonstrates that the IgG1 bias
of a B cell response to infection in DR1 mice can be overridden by
bystander influences of events taking place in the same tissue.
Virus type and host influence innate responses to
To explore whether any differences in innate responses could be
detected in DR1 and B10 mice, we measured the levels of a large
range of cytokines and chemokines in the lungs of DR1 and B10
mice 60 h after infection with influenza NC or PR8 (Fig. 9 and
Fig. S4). We speculated that the levels of proinflammatory and
Th1-polarizing factors would be lower in DR1 compared with B10
mice after NC infection. However, this idea was not supported by
our results. After NC infection, there was a trend towards higher
levels of several cytokines and chemokines in DR1 than in B10
mice (IFN-c and IL-6 for example), but differences were not
statistically significant. Thus, we found no evidence for inherent
differences in early innate responses after NC infection of DR1
Figure 4. Serum levels of virus-specific Ab in DR1 and B10 mice after NC infection. NC-specific IgM (A), IgG1 (B), IgG2b (C), IgG2c (D), IgG3
(E), IgA (F), IgG (G), and Ig (H) titers were determined by ELISA on plates coated with disrupted viral particles. Titers are shown as the reciprocal of the
highest serum dilution scored as positive relative to naı ¨ve control serum. The mean+SE is shown for 4–8 individual mice per group. * P,0.05,
Modulation of T and B cell Responses to Influenza
PLoS ONE | www.plosone.org6 March 2012 | Volume 7 | Issue 3 | e34377
and B10 mice that would explain the distinct patterns of CD4 T cell
cytokines and B cell responses. The clear trend after PR8 infection
was a greater increase in the baseline levels of proinflammatory
factors in DR1 than in B10 mice. IFN-c was the only measured
factor with a concentration that was significantly higher than
baseline levels after PR8 infection of B10 mice, whereas the levels of
multiple proinflammatory cytokines and chemokines (IFN-a, IFN-
c, TNF-a, IL-1a, IL-6, MCP-1, MIP-1b, MIP-2, and IP-10 for
example) were significantly increased in DR1 mice. Interestingly,
there were also significant increases in the concentrations of the
Th2-type cytokines IL-4 and IL-5 in DR1 mice after PR8 infection.
Taken together, our findings raise the possibility that the
concentrations of proinflammatory factors in the lungs of DR1
mice after PR8 infection were sufficient to override influences that
predominated after NC infection.
The current study was initiated to relate patterns of cytokine
secretion by influenza-specific CD4 T cells to epitope specificity
Figure 5. The virus-specific B cell and CD4 T cell response to intramuscular immunization. (A–D) Virus-specific ASC frequencies. DR1 (A
and B) and B10 (C and D) mice were immunized intramuscularly with inactivated NC. Virus-specific ASC frequencies in the iliac lymph nodes and
spleen were determined by ELISpot assay at the indicated times after immunization. The mean+SE is shown for 3–4 individual mice per group. (E, F)
Cytokine production by peptide-specific CD4 T cells. Enriched CD4 T cells from the spleen were analyzed on day 8 after immunization. Frequencies of
CD4 T cells secreting IL-2, IFN-c, or IL-4 were determined by ELISpot assay after in vitro stimulation with antigen-presenting cells and sets of 1–3 17-
mer peptides from different viral proteins (HA, NA, NP, and M1). Results are normalized to spot counts per 106CD4 T cells and are shown as the mean
6 range for 2 independent experiments (the M1 protein was represented in only one experiment with B10 mice). Cells from at least three mice were
pooled for each experiment. (G, H) Proportions of peptide-specific CD4 T cells secreting IL-2, IFN-c, or IL-4. Results are compiled from the data shown
in E and F. The mean 6 range is shown for the two independent experiments.
Modulation of T and B cell Responses to Influenza
PLoS ONE | www.plosone.org7 March 2012 | Volume 7 | Issue 3 | e34377
and the magnitude of epitope-specific responses. A comprehensive
set of epitopes in DR1 and B10 mice were available to us, since we
had recently analyzed these mice after infection with influenza NC
and defined MHC II-restricted epitopes in a broad range of viral
proteins [13,14,21]. The epitopes defined in DR1 mice were
HLA-DR1-restricted, whereas those in B10 were restricted to I-
Ab, the only MHC II molecule expressed in this strain. DR1 mice
represent an established transgenic system for defining HLA-DR1-
restricted epitopes that may be relevant to human responses and
for functional studies of the epitope-specific CD4 T cells [17,25].
Antigen-presenting cells in DR1 mice express a transgene-encoded
hybrid MHC II molecule consisting of the peptide-binding domain
of human HLA-DR1 and a membrane-proximal region of mouse
origin, a structure that permits normal interaction of the
transgenic molecule with murine CD4 during T cell epitope
recognition. The repertoire that we defined in DR1 mice was
exceptionally broad and included multiple strong epitopes in all of
the viral proteins examined (HA, NA, NP, M1, and NS1). In
contrast, the repertoire in B10 mice was much narrower and
comprised approximately one-fifth the number of epitopes in DR1
mice. Within the same set of viral proteins in the B10 study, strong
or moderate epitopes were confined to the NP and NA. Both DR1
and B10 mice were evaluated in the current study, which screened
epitope-specific CD4 T cells for the secretion of IL-2 (the cytokine
used as readout during epitope identification), as well as IFN-c and
IL-4, the prototypic Th1- and Th2-type cytokines, respectively. An
observation that was consistent in DR1 and B10 mice after NC
infection was that the pattern of secretion of IL-2, IFN-c, and IL-4
was independent of epitope specificity or whether epitopes were
major or minor in terms of the number of stimulated CD4 T cells.
Apparently, the priming environment is the overriding influence
on the quality of the CD4 T cell response. What also emerged
from our analysis after NC infection were differences in cytokine
secretion patterns between DR1 and B10 mice. This prompted us
to evaluate CD4 T cell and B cell responses in parallel because of
the link between CD4 T cell-derived cytokines and the pattern of
Ig isotype expression.
A clear majority among all epitope-specific CD4 T cells in the
MedLN and spleen of B10 mice after NC infection secreted IFN-c,
consistent with previous studies and the generally accepted Th1
nature of the response to influenza infection [9,10]. The MedLN
differed from the spleen in having a higher ratio of IFN-c-secreting
to IL-4-secreting CD4 T cells. Our analysis does not identify
whether epitope-specific CD4 T cells produced one or more of the
Figure 6. The B cell response following infection with different viruses that replicate in the lung. (A–F) Virus-specific ASC frequencies.
DR1 (A–C) and B10 (D–F) mice were infected intranasally with the influenza viruses PR8 (A and D) and X31 (B and E), and with the non-influenza virus
MHV68 (C and F). Virus-specific ASC frequencies in the MedLN were determined by ELISPOT assay at intervals after infection. (G) PR8 replication in the
lung. Titers are represented as log10TCID50/0.2 ml of lung homogenate. (H, I) Flow cytometric analysis of ASCs and germinal center B cells in the
MedLN on day 8 after PR8 infection. ASC frequencies (H) represent the proportion of B220intCD138+cells after gating on live CD42CD82CD19+cells.
Germinal center B cell frequencies (I) represent the proportion of PNA+Fas+cells among live CD42CD82B220+cells. (J) Serum levels of virus-specific
IgG in DR1 and B10 mice on day 8 after PR8 infection. Titers determined by ELISA are shown as the reciprocal of the highest serum dilution scored as
positive relative to naı ¨ve control serum. Fig. 6 data sets depict the mean+SE for 3–10 individual mice per group. * P,0.05, *** P,0.001.
Modulation of T and B cell Responses to Influenza
PLoS ONE | www.plosone.org8 March 2012 | Volume 7 | Issue 3 | e34377
cytokines tested. The majority of IL-2-secreting cells in a Th1-
polarized response are also likely to secrete IFN-c [26,27]. Our
observation of a marked predominance of IFN-c-secreting over
IL-2-secreting cells in the B10 response is consistent with an
expected subset of IFN-c-secreting cells that are IL-2-negative
. In all likelihood the IFN-c-secreting and IL-4-secreting cells
represent distinct populations . IFN-c is strongly associated
with the expression of IgG2a/c and IgG2b by activated B cells,
whereas IL-4 negatively regulates production of these isotypes and
promotes IgG1 expression . Thus, the cytokine secretion
patterns fit well with the prominence of IgG2b and IgG2c in the
NC-specific Ab response, especially in the MedLN, and the larger
proportion of IgG1 ASCs in the spleen. It is unclear whether the
different patterns of cytokine secretion by CD4 T cells in the
MedLN compared with the spleen reflect different polarizing
influences on cells activated in the two sites and/or differences
between Th cell subsets in site-to-site trafficking after activation.
The pattern of cytokine production by epitope-specific CD4 T
cells in DR1 mice after NC infection was surprising. In contrast to
the Th1-polarized response and IFN-c predominance in B10
mice, IL-4 secretion was prominent and comparable to IFN-c
secretion in both the MedLN and spleen of DR1 mice. In
addition, the NC-specific ASC response in DR1 mice had unusual
features and differed from what can be considered a typical B cell
response as exemplified in B10 mice [6,29]. Although early IgM
ASC formation was similar in DR1 and B10 mice, the initial
IgG2b/IgG2c response in B10 was essentially absent in DR1 mice.
When an IgG response developed in DR1 mice, it was strongly
IgG1-biased. The skewing towards IgG1 production in DR1 mice
is consistent with the lower ratio of IFN-c-secreting to IL-4-
secreting CD4 T cells. This is further supported by the similarity
between the IgG isotype distribution pattern in DR1 mice and the
response to influenza in IFN-c-deficient mouse models [30,31].
The prominence of IL-4-secreting CD4 T cells in DR1 mice also
suggests an explanation for the stronger GC reaction that we
demonstrated in these mice compared with B10 mice. We did not
measure Tfh cell frequencies in our analysis. However, there is
evidence that IL-4 secretion in responding lymph nodes is largely
restricted to Tfh cells [32,33], suggesting that these cells may be
more frequent in DR1 than in B10 mice. If this were the case, a
stronger GC reaction in DR1 mice would fit with evidence for a
direct relationship between the numbers of Tfh cells and GC B
cells . A recent study indicated that selection of B cells for
entry into the GC reaction is favored by a high level of
peptide:MHC II expression and the advantage conferred in
competition for potentially limiting cognate T cell help .
Conceivably, T cell help to drive GC B cell formation may be less
limiting in DR1 than in B10 mice because of the substantially
broader CD4 T cell repertoire in DR1 mice [13,14,21]. This may
contribute to the greater number of Tfh cells in DR1 mice, since
Tfh cell formation is dependent on signals delivered during
cognate T-B interactions.
The characteristics of the response to i.m. vaccination with
inactivated NC differed from that following NC infection in both
DR1 and B10 mice. In B10 mice, there was a shift in the
predominant CD4 T cell-secreted cytokine from IFN-c after
infection to IL-2 after vaccination. This trend has been described
for CD4 T cells generated by protein vaccines compared with
infections , and likely reflects the influence of infection-
associated innate signals and the induction of factors that drive
Th1 polarization. Although the cytokine secretion pattern in DR1
mice after i.m. vaccination resembled that after infection, the B
cell response differed markedly; notably, the delayed IgG response
and IgG1 bias after infection was not present after vaccination.
Strikingly, B cell responses after vaccination were largely
indistinguishable in DR1 and B10 mice and featured a
predominance of the IFN-c-associated IgG2c among switched
Ab isotypes. Our findings demonstrate that IL-2-biased CD4 T
cell responses generated by protein vaccines can support B cell
responses that are typically associated with IFN-c-secreting Th1-
polarized cells. In addition, they indicate that the differences
between DR1 and B10 mice in responses to lung infection reflect
mechanisms that play little role in responses to i.m. vaccination.
Infection with the influenza viruses PR8 and X31 and the non-
influenza virus MHV68 modulated key characteristics of the B cell
response to NC infection in DR1 mice. Perhaps most notably, the
IgG1 bias that was present in DR1 mice after NC infection was
not present and the distribution of IgG isotypes largely resembled
that in B10 mice. This may reflect an increase in the ratio of IFN-
c-secreting to IL-4-secreting CD4 T cells, as we demonstrated in
DR1 mice after infection with PR8 compared with NC. Our
analysis of early cytokine and chemokine production in the lung
clearly demonstrated the greater capacity of PR8 (compared with
NC) to induce factors that promote a Th1-polarizing environment.
This likely reflects viral virulence determinants in PR8, such as
replicative capacity, cell tropism, and cytotoxic potential, and a
high level of activation of innate responses via pattern recognition
receptor signaling. We demonstrated that co-infection changed the
bias of the IgG response to NC infection in DR1 mice from IgG1
to IgG2b/IgG2c, indicating that this was mediated by non-
specifically acting factors in the priming environment. In addition
to their influence on CD4 T cell responses, innate factors and
pattern recognition receptor ligands may also modulate B cell
Figure 7. The CD4 T cell response to PR8 infection. (A, B)
Cytokine production by peptide-specific CD4 T cells. DR1 and B10 mice
were infected intranasally with PR8. Enriched CD4 T cells from the
MedLN were analyzed on day 10 after infection. Frequencies of CD4 T
cells secreting IL-2, IFN-c, or IL-4 were determined by ELISpot assay after
in vitro stimulation with antigen-presenting cells and individual 17-mer
peptides. Peptide designations (x-axis) include the viral proteins of
origin (NP, M1, and NS1). Results are normalized to spot counts per 106
CD4 T cells and are shown as the mean 6 range for 2 independent
experiments. Cells from at least three mice were pooled for each
experiment. (C, D) Proportions of peptide-specific CD4 T cells secreting
IL-2, IFN-c, or IL-4. Results are compiled from the data shown in A and B.
The mean 6 range is shown for the two independent experiments.
Modulation of T and B cell Responses to Influenza
PLoS ONE | www.plosone.org9 March 2012 | Volume 7 | Issue 3 | e34377
responses through direct interactions with B cells that promote
activation and isotype switching [19,36,37,38].
Two aspects of the early IgG response differed between DR1
and B10 mice after both NC and PR8 infection; virus-specific IgG
production was significantly delayed and the proportion of GC B
cells was significantly higher in DR1 mice. These differences are
apparently unrelated to changes in the CD4 T cell cytokine
secretion patterns that we analyzed. Cognate T cell help is a
prerequisite for B cell entry into the GC reaction . There is
evidence that isotype switching decisions by B cells may be made
before entry into GCs and thus may reflect the cytokine milieu
during cognate T-B interactions . Our observations may
therefore reflect (i) a greater tendency for cognate T-B interactions
to drive GC differentiation of B cells in DR1 mice compared with
B10 mice (as discussed above), and (ii) modulation of CD4 T cell-
secreted cytokine patterns by the type of infecting virus. The delay
in virus-specific IgG production in DR1 mice would be explained
if B cells directed to express any of the IgG1, IgG2b, or IgG2c
isotypes after cognate T-B interactions preferentially differentiated
via the GC pathway instead of rapidly forming ASCs via the
alternative extrafollicular pathway. Interestingly, although IgG
production after PR8 infection was delayed in DR1 compared
with B10 mice, the IgM response was more rapid and substantially
stronger in the DR1 mice. This feature of the IgM response in
DR1 mice was also evident after X31 and MHV68 infection, and
occasionally and to a much lesser degree after NC infection. The
Figure 8. The NC-specific B cell response in DR1 mice is modulated by concurrent infection with MHV68. (A–D) Virus-specific ASC
frequencies. DR1 and B10 mice were infected intranasally with NC only, or with NC and MHV68. ASC frequencies in the MedLN were determined by
ELISPOT assay on day 8 (A and B) and day 10 (C and D) after infection. ASCs specific for NC or MHV68 were detected using plates coated with
disrupted NC (A and C) and disrupted MHV68 (B and D), respectively. (E, F) NC-specific serum IgG (E) and Ig (F) levels. Titers were determined by ELISA
and are shown as the reciprocal of the highest serum dilution scored as positive relative to naı ¨ve control serum. The mean+SE is shown for 4–9
individual mice per group. * P,0.05, ** P,0.01.
Modulation of T and B cell Responses to Influenza
PLoS ONE | www.plosone.org 10 March 2012 | Volume 7 | Issue 3 | e34377
characteristics of the IgM and IgG responses in DR1 mice are
reflected in the markedly higher ratio of virus-specific IgM ASCs
to switched isotype ASCs early in the response in DR1 compared
with B10 mice for all of the viruses used in our analysis (Fig. S5).
The T cell-dependent extrafollicular pathway of B cell
differentiation is thought to generate the initial wave of Ab
produced in response to influenza infection . The idea that this
pathway is responsible for the vigorous early IgM response to PR8
infection in DR1 mice is supported by our demonstration that the
response is T cell-dependent and is associated with the appearance
of a population of cells with the phenotype of extrafollicular ASCs.
Regulation of the extrafollicular pathway is not well understood,
but there is evidence that it is driven, at least in part, by the activity
of innate factors such as type I IFN and IL-12 as well as cytokines
derived from activated T cells [7,36,40,41]. Our analysis of lung
cytokines and chemokines after infection demonstrated a general
pattern of higher levels in DR1 than in B10 mice. In addition, the
levels of a number of these factors were markedly higher in DR1
mice after PR8 than after NC infection. Although many of these
factors have not been investigated in the context of extrafollicular
responses, our observations raise the possibility that the extra-
follicular arm of the B cell response is stronger in DR1 than in B10
mice, perhaps especially after infection with PR8, X31, and
MHV68. Innate factors may also enhance IgM production ,
Figure 9. Cytokine and chemokine production in the lung after influenza infection. DR1 and B10 mice were infected intranasally with NC
or PR8 or were mock-infected with PBS. Mice were sampled 60 h after inoculation. Cytokine and chemokine concentrations in clarified lung
homogenates were determined by Multiplex assay or by sandwich ELISA (IFN-a only). Figure 9 shows the results for a selection of the 30 cytokine and
chemokine determinations. Results for the remaining cytokines and chemokines are shown in figure S4. The mean+SE is shown for 5 individual mice
per group. * P,0.05, ** P,0.01, *** P,0.001.
Modulation of T and B cell Responses to Influenza
PLoS ONE | www.plosone.org11 March 2012 | Volume 7 | Issue 3 | e34377
resulting in an IgM bias in extrafollicular Ab production. A GC
contribution to the early IgM response cannot be excluded, since
GC B cells had developed in the MedLN of PR8-infected DR1
mice at the time of peak IgM ASC numbers.
Our understanding of the B cell response differences between
DR1 and B10 mice is incomplete. Overall, our findings indicate
that these differences are restricted to responses in the respiratory
tract and are modulated (but not eliminated) by the type of
infecting virus through effects on the quality of the CD4 T cell
response. The difference between DR1 and B10 mice was most
distinct after NC infection; DR1 and B10 mice were clearly
different at the level of CD4 T cell cytokine secretion patterns and
the profile of IgG isotype expression after NC infection, but not
after PR8 infection. Although our data are consistent with a
difference that is imparted at the initiation of the response in the
lung, our analysis of early cytokine and chemokine production in
the lungs after NC infection did not suggest basis for this
difference. There is evidence that antigen-presenting cells can be
programmed to bias CD4 T cell responses toward the Th2
pathway of differentiation . An antigen-presenting cell subset
termed late-activator antigen-presenting cells or LAPCs have been
shown to migrate from the lung to the MedLN during influenza
infection and selectively induce Th2 responses . It will be of
interest to evaluate the activity of LAPCs during responses to
different viruses and in different mouse strains.
In summary, our analysis demonstrates that the pattern of
secretion of IL-2, IFN-c, and IL-4 by activated CD4 T cells is
largely independent of epitope specificity and the magnitude of the
epitope-specific response. Rather, the more important influences
on these responses are the form of immunization and inherent host
characteristics. In addition, we demonstrate that CD4 T cell
responses to infection of the respiratory tract are substantially
modulated by the type of infecting virus in a manner that is host-
dependent and reflects changes in the early induction of innate
factors. What also emerges from our analysis is the DR1 mouse as
a potentially useful model to gain insights into regulation of the B
cell response to viral infection of the respiratory tract. An
unexpected but important insight derived from our work is the
importance of immunization strategy for revealing inherent host
differences in CD4 T cell and B cell responses.
germinal center B cells. MedLN cells were analyzed on day 8
after intranasal NC infection of DR1 and B10 mice. Represen-
tative 5% contour plots are shown for individual DR1 and B10
Flow cytometric identification of ASCs and
mice after infection and for naı ¨ve control mice. Plots depict B220
and CD138 expression after gating on live CD42CD82CD19+
cells (A) or PNA-binding and Fas expression after gating on live
CD42CD82B220+cells (B). Numbers indicate cell frequencies in
the depicted gates identifying ASCs (defined as B220intCD138+)
and germinal center B cells (defined as PNA+Fas+).
ed DR1 and B10 mice. Titers were determined by ELISA and
quantified by reference to standards of known concentration. The
mean+SE is shown for 5 individual mice per group.
Total serum levels of Ab isotypes in uninfect-
infection in DR1 mice is CD4 T cell-dependent. PR8-
specific ASC frequencies in the MedLN of CD4 T cell-depleted
DR1 mice and mock-depleted control mice were determined by
ELISpot assay on day 8 after infection. The mean+SE is shown for
4 individual mice per group.
The virus-specific B cell response to PR8
lung after influenza infection. DR1 and B10 mice were
infected intranasally with NC or PR8 or were mock-infected with
PBS. Mice were sampled 60 h after inoculation. Cytokine and
chemokine concentrations in clarified lung homogenates were
determined by Multiplex assay. The mean+SE is shown for 5
individual mice per group. * P,0.05, ** P,0.01, *** P,0.001.
Cytokine and chemokine production in the
isotype ASCs in DR1 and B10 mice after infection with
different viruses. Data were collected from experiments
(presented in figures 3 and 6) in which mice were infected
intranasally with NC, PR8, X31, or MHV68, and virus-specific
ASC frequencies were determined by ELISpot assay. Ratios,
shown for the MedLN on day 8 after infection, were calculated by
dividing the IgM ASC frequency by the sum of the IgG1, IgG2b,
IgG2c, IgG3, and IgA ASC frequencies. The mean ratio+SE is
shown for 4–8 individual mice per group.
Ratio of virus-specific IgM ASCs to switched
Conceived and designed the experiments: DJT A. Sant MYS. Performed
the experiments: A. Sundararajan LH KAR GM SA HJ MYS. Analyzed
the data: HY DJT A. Sant MYS. Contributed reagents/materials/analysis
tools: RJW. Wrote the paper: A. Sant MYS.
1. La Gruta NL, Kedzierska K, Stambas J, Doherty PC (2007) A question of self-
preservation: immunopathology in influenza virus infection. Immunol Cell Biol
Fukuyama S, Kawaoka Y (2011) The pathogenesis of influenza virus infections:
the contributions of virus and host factors. Curr Opin Immunol 23: 481–486.
Iwasaki A, Medzhitov R (2010) Regulation of adaptive immunity by the innate
immune system. Science 327: 291–295.
Waffarn EE, Baumgarth N (2011) Protective B cell responses to flu–no fluke!
J Immunol 186: 3823–3829.
Lee BO, Rangel-Moreno J, Moyron-Quiroz JE, Hartson L, Makris M, et al.
(2005) CD4 T cell-independent antibody response promotes resolution of
primary influenza infection and helps to prevent reinfection. J Immunol 175:
Sangster MY, Riberdy JM, Gonzalez M, Topham DJ, Baumgarth N, et al.
(2003) An early CD4+T cell-dependent immunoglobulin A response to
influenza infection in the absence of key cognate T-B interactions. J Exp Med
Goodnow CC, Vinuesa CG, Randall KL, Mackay F, Brink R (2010) Control
systems and decision making for antibody production. Nat Immunol 11: 681–688.
8. Vinuesa CG, Linterman MA, Goodnow CC, Randall KL (2010) T cells and
follicular dendritic cells in germinal center B-cell formation and selection.
Immunol Rev 237: 72–89.
Sarawar SR, Doherty PC (1994) Concurrent production of interleukin-2,
interleukin-10, and gamma interferon in the regional lymph nodes of mice with
influenza pneumonia. J Virol 68: 3112–3119.
10. Roman E, Miller E, Harmsen A, Wiley J, Von Andrian UH, et al. (2002) CD4
effector T cell subsets in the response to influenza: heterogeneity, migration, and
function. J Exp Med 196: 957–968.
11. Peng SL, Szabo SJ, Glimcher LH (2002) T-bet regulates IgG class switching and
pathogenic autoantibody production. Proc Natl Acad Sci U S A 99: 5545–5550.
12. Stavnezer J (1996) Immunoglobulin class switching. Curr Opin Immunol 8:
13. Richards KA, Chaves FA, Krafcik FR, Topham DJ, Lazarski CA, et al. (2007)
Direct ex vivo analyses of HLA-DR1 transgenic mice reveal an exceptionally
broad pattern of immunodominance in the primary HLA-DR1-restricted CD4
T-cell response to influenza virus hemagglutinin. J Virol 81: 7608–7619.
14. Richards KA, Chaves FA, Sant AJ (2009) Infection of HLA-DR1 transgenic
mice with a human isolate of influenza a virus (H1N1) primes a diverse CD4 T-
Modulation of T and B cell Responses to Influenza
PLoS ONE | www.plosone.org12March 2012 | Volume 7 | Issue 3 | e34377
cell repertoire that includes CD4 T cells with heterosubtypic cross-reactivity to Download full-text
avian (H5N1) influenza virus. J Virol 83: 6566–6577.
15. Joo HM, He Y, Sangster MY (2008) Broad dispersion and lung localization of
virus-specific memory B cells induced by influenza pneumonia. Proc Natl Acad
Sci U S A 105: 3485–3490.
16. Sangster MY, Topham DJ, D’Costa S, Cardin RD, Marion TN, et al. (2000)
Analysis of the virus-specific and nonspecific B cell response to a persistent B-
lymphotropic gammaherpesvirus. J Immunol 164: 1820–1828.
17. Woods A, Chen HY, Trumbauer ME, Sirotina A, Cummings R, et al. (1994)
Human major histocompatibility complex class II-restricted T cell responses in
transgenic mice. J Exp Med 180: 173–181.
18. McDuffie E, Obert L, Chupka J, Sigler R (2006) Detection of cytokine protein
expression in mouse lung homogenates using suspension bead array. J Inflamm
(Lond) 3: 15.
19. Coro ES, Chang WL, Baumgarth N (2006) Type I IFN receptor signals directly
stimulate local B cells early following influenza virus infection. J Immunol 176:
20. Shinall SM, Gonzalez-Fernandez M, Noelle RJ, Waldschmidt TJ (2000)
Identification of murine germinal center B cell subsets defined by the expression
of surface isotypes and differentiation antigens. J Immunol 164: 5729–5738.
21. Nayak JL, Richards KA, Chaves FA, Sant AJ (2010) Analyses of the specificity of
CD4 T cells during the primary immune response to influenza virus reveals
dramatic MHC-linked asymmetries in reactivity to individual viral proteins.
Viral Immunol 23: 169–180.
22. Li X, Vanitha DJ, Joo HM, He Y, Rouse BT, et al. (2006) A strategy for
selective, CD4+T cell-independent activation of virus-specific memory B cells
for limiting dilution analysis. J Immunol Methods 313: 110–118.
23. Joo HM, He Y, Sundararajan A, Huan L, Sangster MY (2010) Quantitative
analysis of influenza virus-specific B cell memory generated by different routes of
inactivated virus vaccination. Vaccine 28: 2186–2194.
24. Szomolanyi-Tsuda E, Welsh RM (1998) T-cell-independent antiviral antibody
responses. Curr Opin Immunol 10: 431–435.
25. Rosloniec EF, Brand DD, Myers LK, Whittington KB, Gumanovskaya M, et al.
(1997) An HLA-DR1 transgene confers susceptibility to collagen-induced
arthritis elicited with human type II collagen. J Exp Med 185: 1113–1122.
26. Divekar AA, Zaiss DM, Lee FE, Liu D, Topham DJ, et al. (2006) Protein
vaccines induce uncommitted IL-2-secreting human and mouse CD4 T cells,
whereas infections induce more IFN-c-secreting cells. J Immunol 176:
27. Wang X, Mosmann T (2001) In vivo priming of CD4 T cells that produce
interleukin (IL)-2 but not IL-4 or interferon (IFN)-c, and can subsequently
differentiate into IL-4- or IFN-c-secreting cells. J Exp Med 194: 1069–1080.
28. Mosmann TR, Cherwinski H, Bond MW, Giedlin MA, Coffman RL (1986)
Two types of murine helper T cell clone. I. Definition according to profiles of
lymphokine activities and secreted proteins. J Immunol 136: 2348–2357.
29. Lipatov AS, Andreansky S, Webby RJ, Hulse DJ, Rehg JE, et al. (2005)
Pathogenesis of Hong Kong H5N1 influenza virus NS gene reassortants in mice:
the role of cytokines and B- and T-cell responses. J Gen Virol 86: 1121–1130.
30. Sarawar SR, Sangster M, Coffman RL, Doherty PC (1994) Administration of
anti-IFN-c antibody to b2-microglobulin-deficient mice delays influenza virus
clearance but does not switch the response to a T helper cell 2 phenotype.
J Immunol 153: 1246–1253.
31. Graham MB, Dalton DK, Giltinan D, Braciale VL, Stewart TA, et al. (1993)
Response to influenza infection in mice with a targeted disruption in the
interferon c gene. J Exp Med 178: 1725–1732.
32. Reinhardt RL, Liang HE, Locksley RM (2009) Cytokine-secreting follicular T
cells shape the antibody repertoire. Nat Immunol 10: 385–393.
33. King IL, Mohrs M (2009) IL-4-producing CD4+T cells in reactive lymph nodes
during helminth infection are T follicular helper cells. J Exp Med 206:
34. Rolf J, Bell SE, Kovesdi D, Janas ML, Soond DR, et al. (2010) Phosphoinositide
3-kinase activity in T cells regulates the magnitude of the germinal center
reaction. J Immunol 185: 4042–4052.
35. Schwickert TA, Victora GD, Fooksman DR, Kamphorst AO, Mugnier MR, et
al. (2011) A dynamic T cell-limited checkpoint regulates affinity-dependent B
cell entry into the germinal center. J Exp Med 208: 1243–1252.
36. Chang WL, Coro ES, Rau FC, Xiao Y, Erle DJ, et al. (2007) Influenza virus
infection causes global respiratory tract B cell response modulation via innate
immune signals. J Immunol 178: 1457–1467.
37. Liu N, Ohnishi N, Ni L, Akira S, Bacon KB (2003) CpG directly induces T-bet
expression and inhibits IgG1 and IgE switching in B cells. Nat Immunol 4:
38. Jegerlehner A, Maurer P, Bessa J, Hinton HJ, Kopf M, et al. (2007) TLR9
signaling in B cells determines class switch recombination to IgG2a. J Immunol
39. Stavnezer J, Guikema JE, Schrader CE (2008) Mechanism and regulation of
class switch recombination. Annu Rev Immunol 26: 261–292.
40. Kim SJ, Caton M, Wang C, Khalil M, Zhou ZJ, et al. (2008) Increased IL-12
inhibits B cells’ differentiation to germinal center cells and promotes
differentiation to short-lived plasmablasts. J Exp Med 205: 2437–2448.
41. Rothaeusler K, Baumgarth N (2010) B-cell fate decisions following influenza
virus infection. Eur J Immunol 40: 366–377.
42. Schmitz N, Kurrer M, Bachmann MF, Kopf M (2005) Interleukin-1 is
responsible for acute lung immunopathology but increases survival of respiratory
influenza virus infection. J Virol 79: 6441–6448.
43. Arima K, Watanabe N, Hanabuchi S, Chang M, Sun SC, et al. (2010) Distinct
signal codes generate dendritic cell functional plasticity. Sci Signal 3: ra4.
44. Yoo JK, Galligan CL, Virtanen C, Fish EN (2010) Identification of a novel
antigen-presenting cell population modulating antiinfluenza type 2 immunity.
J Exp Med 207: 1435–1451.
Modulation of T and B cell Responses to Influenza
PLoS ONE | www.plosone.org13 March 2012 | Volume 7 | Issue 3 | e34377