Download Antigen Responses to a Secondary T-Independent T

This information is current as
of June 17, 2017.
Acute Plasmodium chabaudi Infection
Dampens Humoral Responses to a Secondary
T-Dependent Antigen but Enhances
Responses to a Secondary T-Independent
Antigen
Joel R. Wilmore, Alexander C. Maue, Julie S. Lefebvre,
Laura Haynes and Rosemary Rochford
Subscription
Permissions
Email Alerts
Information about subscribing to The Journal of Immunology is online at:
http://jimmunol.org/subscription
Submit copyright permission requests at:
http://www.aai.org/About/Publications/JI/copyright.html
Receive free email-alerts when new articles cite this article. Sign up at:
http://jimmunol.org/alerts
The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2013 by The American Association of
Immunologists, Inc. All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
J Immunol published online 30 September 2013
http://www.jimmunol.org/content/early/2013/09/30/jimmun
ol.1301450
The Journal of Immunology
Acute Plasmodium chabaudi Infection Dampens Humoral
Responses to a Secondary T-Dependent Antigen but Enhances
Responses to a Secondary T-Independent Antigen
Joel R. Wilmore,*,1 Alexander C. Maue,*,1,2 Julie S. Lefebvre,† Laura Haynes,‡ and
Rosemary Rochford*
I
n many parts of the world, especially in the tropics where
mosquito-borne diseases and parasitic infections are common, there is a high frequency of coinfection (1, 2). Coinfection can lead to an exacerbation of one or both of the diseases
because of the skewing of the immune response away from the
optimal response needed for clearance of infection (3, 4). Globally, there are more than 225 million new cases of malaria each
year with the greatest burden on children under 5 y of age (5). In
malaria endemic regions, there is a significantly higher rate of
“all-cause” morbidity and mortality that is greater than the effect
of increased malaria infection in the population (6), suggesting
*Department of Microbiology and Immunology, State University of New York, Upstate Medical University, Syracuse, NY 13210; †Trudeau Institute, Saranac Lake, NY
12983; and ‡Center on Aging, University of Connecticut Health Center, Farmington,
CT 06030
1
J.R.W. and A.C.M. contributed equally to this work.
2
Current address: Enteric Diseases Department, Naval Medical Research Center,
Silver Spring, MD.
Received for publication March 31, 2013. Accepted for publication September 3,
2013.
This work was supported by a Hendricks postdoctoral research grant from the State
University of New York Upstate Medical University (to A.C.M.), U.S. Department of
Health and Human Services, National Institutes of Health, National Cancer Institute
Grant R01CA102667 (to R.R.), and a postdoctoral fellowship grant from the Fonds
de recherche du Quebec - Santé (to J.S.L.).
Address correspondence and reprint requests to Dr. Rosemary Rochford, Department
of Microbiology and Immunology, State University of New York, Upstate Medical
University, 2237C Weiskotten Hall, Syracuse, NY 13210. E-mail address: rochforr@
upstate.edu
Abbreviations used in this article: Be1, B effector 1; CGG, chicken gamma globulin;
DC, dendritic cell; dpi, day postinfection; GC, germinal center; NP, 4-hydroxy-3nitrophenyl; PNA, peanut lectin (agglutinin); pRBC, parasitized RBC; TD, T-dependent; TI, T-independent.
Copyright Ó 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1301450
a greater impact of malaria than infection alone. In these regions,
it is common for people to have subpatent malaria infection (7),
and therefore, it is important to understand how an established
malaria infection could inhibit immunity to a newly acquired infection or vaccine. Evidence for a role of malaria in affecting
vaccine efficacy is demonstrated by increased mortality of children
living in endemic regions despite increased vaccination coverage
(8). For example, seroconversion rates for the poliovirus vaccine
are reduced by 30% in African children compared with their North
American counterparts (9). In addition, malaria is thought to be
the causative agent behind impaired Ab responses for vaccines such
as tetanus (10), pertussis (10), Haemophilus (11), and typhoid (12).
Understanding the effects of malaria on immunity to secondary
pathogens or vaccines is important to develop treatments that will
diminish the morbidity and mortality caused by high rates of coinfection in malaria endemic regions.
Blood stage malaria infection is systemic, at which time the host
mounts a Th1 response characterized by the production of high
levels of TNF-a following infection with Plasmodium falciparum
(13) and IFN-g following infection with Plasmodium chabaudi
(14). B cells and Ab are crucial for the control of malaria parasites, and clearance of parasitemia requires a switch from the early
Th1 response to a Th2 response as seen from studies done in the
P. chabaudi mouse model (15, 16). The IgG subclass produced in
response to infection can dramatically affect the ability of humoral
immunity to confer protection against disease (17, 18). The Abs
made in response to soluble Plasmodium vaccine candidate proteins are heavily skewed toward the IgG3 isotype in humans (19,
20), which is equivalent to IgG2b in mice (21). In most experimental mouse models of Plasmodium infection, there is a heavy
skewing toward an IgG2a or IgG2c isotype during an in vivo infection (22). IgG2c production is highly associated with increased
levels of IFN-g and decreased levels of IL-4 (23).
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
High rates of coinfection occur in malaria endemic regions, leading to more severe disease outcomes. Understanding how coinfecting
pathogens influence the immune system is important in the development of treatment strategies that reduce morbidity and mortality. Using the Plasmodium chabaudi mouse model of malaria and immunization with model Ags that are either T-dependent (4hydroxy-3-nitrophenyl [NP]-OVA) or T-independent (NP-Ficoll), we analyzed the effects of acute malaria on the development of
humoral immunity to secondary Ags. Total Ig and IgG1 NP–specific Ab responses to NP-OVA were significantly decreased in the
P. chabaudi–infected group compared with the uninfected group, whereas NP-specific IgG2c Ab was significantly increased in the
P. chabaudi–infected group. In contrast, following injection with T-independent NP-Ficoll, the P. chabaudi–infected group had
significantly increased NP-specific total Ig, IgM, and IgG2c Ab titers compared with controls. Treatment with anti–IFN-g led to an
abrogation of the NP-specific IgG2c Ab induced by P. chabaudi infection but did not affect other NP-specific Ab isotypes or titers.
IFN-g depletion also increased the percentage of plasma cells in both P. chabaudi–infected and uninfected groups but decreased
the percentage of B cells with a germinal center (GC) phenotype. Using immunofluorescent microscopy, we were able to detect
NP+ GCs in the spleens of noninfected mice, but there were no detectible NP+ GCs in mice infected with P. chabaudi. These data
suggest that during P. chabaudi infection, there is a shift toward an extrafollicular Ab response that could be responsible for
decreased Ab responses to secondary T-dependent Ags. The Journal of Immunology, 2013, 191: 000–000.
2
MALARIA AFFECTS HUMORAL RESPONSES TO SECONDARY Ag
Materials and Methods
Animals/ethics statement
Male C57BL/6J mice (8–10 wk old) were obtained from The Jackson Laboratory (Bar Harbor, ME) or bred at State University of New York (SUNY)
Upstate Medical University. Animals were maintained under specific
pathogen-free conditions. All research involving animals has been conducted as required by the Animal Welfare Act, Public Health Service
Guidelines, and New York State regulations with respect to husbandry, experimentation, and welfare. Mouse protocols were approved by the SUNY
Upstate Medical University Committee on the Humane Use of Animals.
Experimental P. chabaudi infection and Ag injections
P. chabaudi chabaudi AS was a gift from Dr. J. Stoute (Pennsylvania State
University, Hershey, PA). Parasitized RBCs (pRBCs) were stored at 280˚C
in glycerolyte solution until use. Prior to experimental use, frozen-infected
RBCs were thawed and injected into donor mice for one passage to
propagate P. chabaudi. Naive C57BL/6 mice were injected i.p. with 5 3
105 pRBCs. Parasitemia was determined at selected days postinfection by
microscopic examination of Giemsa-stained thin blood smears.
C57BL/6J mice were injected i.p. with 50 mg NP-OVA adsorbed onto
alum or 10 mg NP-Ficoll (Biosearch Technologies, Novato, CA). NP conjugates were prepared as described previously (26). To determine the acute
effects of P. chabaudi infection on secondary Ag responses, mice were injected 5 d postinfection (dpi). The effects of a prior infection on secondary
Ag were determined by injecting mice 30 d following infection with
P. chabaudi. IFN-g depletion was performed by retro-orbital i.v. injection
of rat anti–IFN-g Ab (clone R4-6A2; BioXCell, West Lebanon, NH) or 1 mg
control rat IgG1 (Jackson ImmunoResearch Laboratories, West Grove, PA)
at 4, 5, 9, and 13 dpi with P. chabaudi.
ELISA
Sera were collected from mice at indicated time-points and assayed by
ELISA to determine NP-specific Ab responses. Briefly, ELISA plates (Nunc,
Rochester, NY) were coated with NP-BSA in carbonate coating buffer (pH
9.6) and blocked with 1% BSA (Sigma-Aldrich, St. Louis, MO) in PBS
containing 0.05% Tween 20 (Sigma-Aldrich). Total Ig, IgG1, IgG3, IgG2b,
and IgG2c NP-specific responses were detected using appropriate peroxidaseconjugated goat anti-mouse Abs (Southern Biotechnology Associates,
Birmingham, AL) and developed using 3,39,5,59-tetramethylbenzidine substrate (eBioscience, San Diego, CA). The reaction was stopped by addition
of 1 M H3PO4 (Sigma-Aldrich), and the absorbance was read at 450nm.
Histology
Spleens were harvested from mice and placed in a mold containing OCT
compound (Sakura, Torrance, CA). The tissues were immediately frozen
over liquid nitrogen and kept at 280˚C until used. Frozen tissue blocks
were brought to 218˚C, and thin cross-sections (7 mm) were cut in a
cryostat (Leica CM1850; Leica Microsystems, Buffalo Grove, IL) and
placed onto poly-L-lysine–coated slides. Slides were dried at room temperature for ∼1–2 h and stored at 280˚C until used. Slides were brought to
room temperature, fixed in acetone/ethanol (75/25, v/v) for 10 min, and
washed three times in PBS to remove the OCT. Slides were then blocked
with 10 mg/ml purified Fc block Ab (BioXCell) in PBS containing 5%
serum (normal mouse serum and normal donkey serum [1:1]) for 30 min
before being probed with FITC-conjugated GL-7 (10 mg/ml; BD Biosciences, San Jose, CA) and NP-allophycocyanin (0.1 mg/ml) in a humidified chamber for 30 min. After washing three times with PBS, slides were
incubated with Alexa Fluor 488–conjugated anti-FITC (20 mg/ml; Invitrogen, Grand Island, NY) for an additional 30 min. Slides were washed
again three times with PBS and mounted with AquaMount (PolySciences
Warrington, PA). Images were captured with a 320 dry objective using
a Leica TCS SP5II confocal microscope and analyzed using the Leica LAS
AF software version 2.2.1 (Leica Microsystems). Contrast and brightness
were adjusted using the FiJi software.
Lymphocyte isolation and flow cytometry
Spleens were aseptically collected from infected and/or immunized mice
at various times. Spleens were mechanically dissociated in PBS and passed
through a 70-mm nylon mesh screen yielding a single-cell suspension. RBCs
were lysed by addition of ammonium chloride lysis buffer. Total and live
cells were enumerated using trypan blue (Sigma-Aldrich) exclusion with a
hemacytometer. Cells were stained in PBS containing 1% BSA and 0.1%
sodium azide on ice with mAbs against the following cell surface molecules:
AA4.1, B220, IgM, CD38 (eBioscience), CD19, CD23, CD21, (BioLegend,
San Jose, CA), and CD138 (BD Biosciences, San Diego, CA). FcRs were
blocked using 2.4G2 mAb. Peanut lectin (agglutinin) (PNA)-FITC (Vector
Laboratories, Burlingame, CA) was used to identify germinal center (GC) phenotype B cells. Cells undergoing apoptosis were determined using an
AnnexinV staining kit (BD Biosciences), according to the manufacturer’s
instructions. NP-binding cells were visualized using NP-allophycocyanin
as previously demonstrated (32) or NP-PE (Biosearch). Following staining,
cells were analyzed using a LSRII analyzer (BD Biosciences) capable of
10-color analysis. Data were analyzed using FlowJo software (Tree Star,
Ashland, OR).
Results
Acute P. chabaudi infection leads to decreased Ab titers to
a secondary NP-OVA immunization and a shift in NP-specific
Ab isotype
To determine whether P. chabaudi infection impacts the generation of Ab to secondary Ags, we evaluated the humoral response
to NP-OVA. This approach allowed us to evaluate Ab responses to
a single, well-defined TD immunogen. We first evaluated whether
the timing of the injection of NP-OVA relative to the stage of
P. chabaudi infection altered the humoral response to NP-OVA.
C57BL/6 mice were infected with P. chabaudi by injecting 5 3
105 pRBCs, and then, mice were injected with the TD Ag NPOVA alone or during acute P. chaubadi infection at 5 dpi or during
resolved infection at 30 dpi. Giemsa-stained blood smears were
used to determine the parasitemia. At 5 dpi, the parasitemia was
on average 12% infected RBC, and at 30 dpi, the parasitemia
ranged from 0.01 to 0.5% infected RBC. Serum was collected to
assess the production of NP-specific Ab by ELISA 12 d post–NPOVA injection to examine the TD response after the initiation of
the GC response. Mice injected with NP-OVA during the acute
phase of P. chabaudi infection exhibited significant reductions
in the production of NP-specific total Ig (H+L) Abs compared
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
Coinfection models are complex, and the effects of immune
system dysregulation can be difficult to differentiate from the effects of the interactions between the infecting agents. The humoral
immune response against 4-hydroxy-3-nitrophenyl (NP) acetylhapten coupled to carrier protein or polysaccharide has been extensively studied and well characterized (24). The use of NPcarrier Ag to mimic a coinfection response during malaria has the
advantages of a lack of competition for resources between the
infecting agents, greater reproducibility between experiments, and
the ability to use widely available reagents to monitor the immune
response to the NP-hapten. The prototypical response to soluble
protein or carbohydrate Ags are of the IgG1 or IgG3 subclasses
in mouse models (25). The predominant Ab isotype made during
immune responses to the model Ag NP-OVA is of the IgG1 subclass (26). High levels of IL-4 and low levels of IFN-g characterize
Th2 responses that are associated with the NP-OVA response (27,
28).
Plasmodium chabaudi chabaudi AS. is a natural rodent pathogen that allows for the study of the blood stage of malaria in
C57BL/6 mice (29). Previous studies using the P. chabaudi model
of malaria infection in mice demonstrate that Ab responses to
a secondary unrelated Ag (e.g., of NP-chicken gamma globulin
[CGG]) are of a lower avidity when injection occurred at the same
time as infection and that P. chabaudi infection had no effect on an
already established humoral memory response to the secondary
Ag (30, 31). These studies examined the effects of P. chabaudi on
secondary injection of Ag prior to the establishment of infection
but not during acute infection. In this study, we analyzed the humoral
immune response to model T-dependent (TD) or T-independent (TI)
secondary Ags during acute P. chabaudi chabaudi (AS) infection.
Our findings suggest that P. chabaudi shifts immunity to secondary
Ags toward predominantly extrafollicular humoral responses.
The Journal of Immunology
with noninfected NP-OVA–injected mice (Fig. 1A). In contrast,
mice infected with P. chabaudi for 30 d prior to NP-OVA injection
had similar levels of NP-specific Abs compared with mice injected
with only NP-OVA (Fig. 1A). The lack of an effect when NP-OVA
injection was given at 30 dpi suggests that the acute phase of
infection plays a crucial role in diminishing the NP-specific Ab
response. In addition, previous studies have shown that there is no
effect if secondary Ag is injected before or at the same time as
secondary Ag (30, 31). Therefore, we focused on the 5 dpi secondary immunization time point throughout the rest of our study.
We next examined the NP-specific IgG isotypes induced following
injection during acute P. chabaudi infection. We observed that NPspecific IgG1 Ab titers were significantly lower in the P. chabaudi–
infected mice compared with uninfected NP-OVA–injected
mice. Levels of NP-specific IgG2b and IgG3 were unaffected by
P. chabaudi infection (Fig. 1B). In addition, although NP-specific
total Ig was reduced, P. chabaudi–infected NP-OVA–injected mice
had higher levels of NP-specific IgG2c in their sera compared with
uninfected NP-OVA–injected animals (Fig. 1B). The increased
3
NP-specific IgG2c Ab production following NP-OVA injection of
P. chabaudi–infected mice suggests that the defect in total Ig may
be primarily a result of a loss of the IgG1 response.
To determine whether the reduction in NP-specific Ab was transient or sustained, we injected control and P. chabaudi–infected mice
with NP-OVA at 5 dpi and examined serum Ab titers at 90 dpi.
There was still a significant reduction in NP-specific total Ig at
90 dpi compared with levels of NP-OVA Ig in mice injected with
NP-OVA alone (Fig. 1C). These data indicate that TD secondary Ag
exposure during acute P. chabaudi infection resulted in significantly
reduced Ab responses at day 17 and that those reduced levels were
sustained out to 90 dpi.
P. chabaudi infection enhances the Ab response to the TI Ag
NP-Ficoll
IFN-g depletion abrogates NP-specific IgG2c production
P. chabaudi induces high levels of serum IFN-g, which skew toward an IgG2c response (35), suggesting that the observed increase in IgG2c was a cytokine-mediated effect. To test whether
IFN-g was necessary for the skewing of the NP-OVA response
from IgG1 to IgG2c, we depleted the cytokine using anti–IFN-g
Abs. P. chabaudi–infected mice were injected with pRBCs at day
0, and then, control and infected mice were immunized with NPOVA/alum at 5 dpi. Anti–IFN-g Ab or control rat IgG was given
i.v. at 4, 5, 9, and 13 dpi. On day 17, ELISAs were performed to
measure NP-specific Ab titers in serum. Depletion of IFN-g did
not affect the levels of NP-specific total Ig (Fig. 3A), IgG1 (Fig.
3B), or IgM (Fig. 3D) when compared with controls. However,
IFN-g depletion did result in significantly reduced NP-specific
IgG2c in the P. chabaudi–infected mice such that the levels were
similar to the uninfected group (Fig. 3C). These results suggest
that IgG2c skewing occurs as a result of the increased serum IFN-g
levels and that IFN-g was not responsible for decreasing the total
Ig and IgG1 NP–specific responses.
FIGURE 1. NP-OVA injection 5 d post–P. chabaudi infection leads
to decreased serum titers of NP-specific Abs and altered NP-specific Ab
isotypes. (A) Serum Ab titers of NP-specific total Ig when NP-OVA was
injected alone (N) or after P. chabaudi infection (n). NP-OVA/alum was
injected at 5 or 30 d post–P. chabaudi, and then, serum was obtained at 12 d
post–NP-OVA. (B) Serum Ab titers for NP-specific IgM, IgG1, IgG2b,
IgG2c, and IgG3 isotypes from mice injected with NP-OVA/alum 5 d post–
P. chabaudi. (C) Total serum NP-specific Ig titers from mice injected with
NP-OVA/alum 5 d post–P. chabaudi and sacrificed 90 d post–NP-OVA injection. (A–C) Representative of two experiments with an n = 4/group [n = 3
of NP alone group in (C)] *p , 0.05 determined by a Mann–Whitney U test.
FIGURE 2. NP-specific Ab titers are enhanced by P. chabaudi when mice
are injected with the TI Ag NP-Ficoll. Serum Ab titers of NP-specific total Ig,
IgM, IgG1, and IgG2c isotypes 7 d postinjection with NP-Ficoll. Mice were
injected with NP-Ficoll alone (d) or NP-Ficoll 5 d post–P. chabaudi infection (N). n = 4/group. *p , 0.05 determined by a Mann–Whitney U test.
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
The next set of experiments addressed whether P. chabaudi infection had any impact on the humoral response to a TI Ag such as
NP-Ficoll (33). Mice were infected with P. chabaudi and at 5 dpi
injected with NP-Ficoll. NP-specific serum Ab titers were determined 7 d post–NP-Ficoll injection to examine the TI immune
response at its peak and before potential contributions from follicular B cells (34). P. chabaudi infection significantly increased
the serum NP-specific Ab titers for total Ig, IgM, IgG2c Abs, and
to a lesser degree IgG1 (Fig. 2). These results suggest that P.
chabaudi infection enhances a TI Ab response while simultaneously suppressing a TD Ab response.
4
MALARIA AFFECTS HUMORAL RESPONSES TO SECONDARY Ag
The percentage and total number of NP-specific B cells is
significantly lower in spleens of P. chabaudi–infected mice
To determine whether the reduced NP response in P. chabaudi–
infected groups was the result of a decrease in NP-specific B cells,
we assayed the percentage of NP+ B cells by flow cytometry. In
addition, we examined the effects of IFN-g depletion on the ability
to produce NP+ B cells at 17 dpi. The total number of splenocytes
isolated from P. chabaudi–infected mice was significantly higher
in all treatment groups compared with NP-OVA alone controls
(Fig. 4B). The percentage and total number of NP+CD19+ B cells
in the total lymphocyte population in the NP-OVA alone group
was significantly higher when compared with the group immunized with NP-OVA during acute P. chabaudi infection in both the
rat IgG control and anti–IFN-g groups (Fig. 4A, 4C). The depletion of IFN-g did not lead to significant differences in NP+ B cells
in the P. chabaudi or uninfected NP-OVA alone mice (Fig. 4A,
4C).
To determine whether reduced NP-specific Ab during P. chabaudi
infection is the result of diminished differentiation of responding
B cells to plasma cells and GC B cells, we examined the phenotype of cells within the spleen. The low percentage of NP+ B cells
restricted our ability to determine whether there were NP+ GC
B cells during P. chabaudi infection. Interestingly, the IFN-g depletion significantly reduced the percentage of GC phenotype
B cells in the spleen as defined by CD19+CD932CD382PNA+
cells in both the NP-OVA alone and NP-OVA and P. chabaudi
(Fig. 4A, 4E), but a large percentage of CD19+CD932CD382PNA2
cells were present. The percentage of plasma cells was determined
by gating on CD138+ cells. In addition, the depletion of IFN-g led
to significantly higher plasma cell percentages in both the NP-OVA
alone and P. chabaudi– and NP-OVA–treated mice, but the total
number of plasma cells was significantly higher in the P. chabaudi–
infected groups for both rat IgG and IFN-g–depleted mice (Fig.
4A, 4D). These results suggest that increased IFN-g resulting
from P. chabaudi infection is driving responding B cells toward
GC differentiation rather than toward the generation of plasma
cells.
NP-OVA injection during acute P. chabaudi infection results in
there being no NP+ cells in GCs
To determine whether P. chabaudi infection influenced the development of NP-specific B cells, we used immunohistochemistry to
examine the localization of NP+ B cells in the spleens of NP-OVA–
injected mice. Fig. 5 shows that uninfected mice immunized with
NP-OVA have NP+ cells present in the spleen that are colocalized
with GL7+ GCs. In contrast, Plasmodium-infected mice immunized with NP-OVA possessed GL7+ GCs, but no GC-associated
or extrafollicular NP+ cells were detected at 17 dpi. These data
suggest the NP-specific GC response has been altered by P. chabaudi
infection.
FIGURE 3. IFN-g depletion decreases the IgG2c response to NP-OVA
that is induced by P. chabaudi, but other Ab isotypes are unaffected. Serum
NP-specific total Ig Ab (A), IgG1 (B), IgG2c (C), and IgM (D) titers for
mice that were untreated, given P. chabaudi alone, NP-OVA/alum alone,
NP-OVA and P. chabaudi, or NP-OVA alone and P. chabaudi + NP-OVA in
the presence of anti–IFN-g Abs or control rat IgG. n = 5 mice/group. *p ,
0.05, **p , 0.001 determined by a Mann–Whitney U test.
Discussion
Coinfections can often result in suboptimal responses to one or
both of the pathogens involved (3, 4). Malaria is a systemic infection that results in a heavy skewing toward a proinflammatory Th1
environment (15). Using the P. chabaudi mouse model of infection, we tested the hypothesis that acute infection with P. chabaudi
would alter the development of humoral immunity to model Ags.
Our results demonstrate that the TD Ab response to NP was significantly reduced with regard to both total Ig and IgG1 Ab levels
typically seen following injection with NP-OVA, whereas the NP-
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
IFN-g depletion leads to increased plasma cell production and
decreased GC B cells
The Journal of Immunology
5
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
FIGURE 4. NP-OVA injection during acute P. chabaudi infection leads to reduced NP+ B cells and lower plasma cells percentages compared with NP-OVA
alone. IFN-g depletion rescues and enhances the plasma cell percentages in P. chabaudi–infected mice and leads to a reduction in GC B cells. (A) Representative flow cytometry of splenic cells taken from mice that were treated with NP-OVA/alum alone or NP-OVA and P. chabaudi in the presence of anti–IFN-g
Abs or control rat IgG. (B) The total cell number per spleen from each of the groups listed above. (C) The percentage of NP+ cells and total cell number per
spleen as represented in the left column of (A). (D) Percentage of plasma cells of the total live cells and total cell number per spleen as demonstrated in the
middle column of (A). (E) The percentage of mature B cells and total cell number per spleen of GC B cells (PNAhiCD382) of the mature B cells (CD19+CD932)
as represented in the right column of (A). n = 4–5 mice/group. *p , 0.05, **p , 0.001 determined by a Mann–Whitney U test.
specific IgG2c response was increased in an IFN-g–dependent
manner. In contrast, humoral immunity to the TI Ag NP-Ficoll
was enhanced by acute P. chabaudi infection. Thus, the Ab response to a secondary Ag was altered during P. chabaudi infection
in both the magnitude and Ab isotype induced.
In human malaria, it has been demonstrated using passive transfer
of immune serum that Abs are critical in providing protection against
blood stage disease (36). However, there is growing evidence that
B cell responses are dysregulated following infection with Plasmodium. Several studies have shown that following infection, Ab
responses to malarial Ags are short-lived (reviewed in Ref. 37).
Similarly, impaired Ab responses also have been observed for several different vaccines in malaria endemic regions (8–12). Kinyanjui
et al. (38) showed that IgG responses in Kenyan children following
6
MALARIA AFFECTS HUMORAL RESPONSES TO SECONDARY Ag
an acute malaria infection exhibited a reduced half-life compared
with the normal values for this Ab subclass. Furthermore, it has
been shown that Ab levels against merozoite Ags are highest during
periods of high parasite transmission and lowest during times
of low transmission (39, 40). Despite these suboptimal Ab responses, several studies have demonstrated in humans (41) and mouse
models (22) that memory B cell and plasma cell responses develop
in response to infection. Weiss et al. (41) showed that human B cell
memory and long-lived Ab levels expand gradually with repeated
infections. In mice, although some studies have shown normal
development of memory B cell responses during Plasmodium infection (22, 42), others have shown defects in memory B cell generation or maintenance (43, 44). Crompton and colleagues (45) also
have suggested that P. falciparum drives short-lived plasma cell
responses, which may explain why Ab levels are not maintained in
the absence of repeated exposures, and because memory B cell
and plasma cell responses are regulated independently of each other,
the presence of memory B cells may not be required to maintain
serum Ab levels.
The predominant IgG subclass induced during an infection can
be critical to the generation of an effective immune response against
that pathogen. Immunization with NP-OVA in alum produces predominately IgG1, which is associated with a strong Th2 response
(27). Our data showed that P. chabaudi infection resulted in a shift
in the NP-specific Ab response from IgG1 to IgG2c. Of potential
significance is that P. chabaudi–specific Abs have been shown to
be predominantly of the IgG2c isotype in response to infection
(22), thus suggesting that P. chabaudi infection resulted in skewing
of the immune response to the secondary Ag. The increase in
plasma cells in response to P. chabaudi in the presence of anti–
IFN-g Ab suggests that the Th1 program induced during infection
is involved in the lack of a long-lived plasma cell response to
secondary Ag. The IgG2c skewing and lack of long-lived plasma
cells also may be indicative of TD extrafollicular responses, such
as those reported against Salmonella (46) where high-affinity IgG2c
was produced in the absence of GCs.
The skewing of IgG isotypes is dependent on the cytokines produced during infection (15). The production of IgG2c (IgG2a in
BALB/c mice) is largely dependent on IFN-g (47). High levels of
IFN-g have also been associated with depressed B cell responses
and lower IgG1 Ab production (47). Our data demonstrate that
when IFN-g is depleted, there is a reduction in NP-specific IgG2c
levels in infected mice. However, IFN-g did not appear to affect
the NP-specific total Ig or IgG1 titer compared with mice injected
with control IgG instead of anti–IFN-g. This suggests that IFN-g
produced during P. chabaudi infection is primarily skewing toward an increase in IgG2c but not inhibiting IgG1 because of there
being no other significant effect on the production of Ab against
secondary Ags following IFN-g depletion. Interestingly, type I IFN
previously has been shown to skew follicular B cell Ab responses
against TI-2 Ags toward an IgG2c isotype (48). Type I IFN is produced during Plasmodium infection (49) and permits B cells to become responsive to IL-12 and become B effector 1 (Be1) cells (50).
Sustained expression of IFN-g and T-bet by Be1 cells is maintained
through a signaling loop involving dendritic cells (DCs) producing
IL-12 and IL-18 leading to expression of IFN-g by Th1 cells and
B cells (23, 51). Our findings together with these studies suggest
that the Th1 cytokine program, as well as potential contributions from
Be1 cells, is a critical component in instructing class switching to
IgG2c.
The timing of P. chabaudi infection and exposure to secondary
Ag was found to be an important factor as demonstrated by significant differences in NP-OVA responses only when injected during acute P. chabaudi infection but not when given at the same
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
FIGURE 5. Splenic GCs do not contain NP+ cells in mice injected with NP-OVA 5 d post–P. chabaudi infection. Spleens from mice injected with NPOVA/alum alone or NP-OVA/alum 5 d post–P. chabaudi infection were removed 12 d post–NP-OVA injection. Spleens were stained with GL7 Ab (green)
for GC B cells and NP-allophycocyanin (red) for NP-specific B cells. Shown are representative images from two mice per group and a naive unimmunized/
uninfected mouse (top panels, NP-OVA alone; bottom panels, P. chabaudi + NP-OVA) of a total of four spleens sectioned per group. Scale bars, 500 mm
(left panels); 100 mm (right panels).
The Journal of Immunology
responses. This work did not examine the effects of Plasmodium
infection on T cell helper functions, which may ultimately affect
the fitness of Ag-specific B cells and their ability to mount an
effective humoral response. T cell function during Plasmodium
infection has been studied in the context of TCR transgenic CD4
T cells specific for merozoite surface protein 1, which proliferated
poorly near the peak of parasitemia (65). In addition, DCs isolated
from the spleens of infected mice have been shown to transiently
lose their ability to activate T cells (65), but timing is important
because other groups have shown DC function is normal in
Plasmodium-infected mice (66). Furthermore, human studies have
demonstrated that CD4 T cells isolated from children with
malaria express cell surface markers associated with exhaustion
(67). Using our experimental model, future studies using NP-OVA as
the experimental Ag will allow for the use of MHC class II tetramers
to analyze the Th cells responding to OVA and further assess the
effect of Plasmodium on the T cell response to NP-OVA immunization. Further work will need to be conducted to assess the
contributions of Ag-presenting and T cells in this experimental
model system.
In sum, the results of this study suggest that during an acute
malaria infection, the Ab responses to secondary Ags/pathogens
will be skewed toward an extrafollicular response that lead to
decreased Ab titers or shift in the predominant Ab isotypes. The
consequence of P. chabaudi infection to the host then would be
a decreased Ab titer and shift in Ab isotype to a secondary Ag that
could lead to a diminished protection. Understanding the weakened humoral immunity to secondary pathogens in malaria endemic regions is important to decrease the morbidity and mortality
resulting from Plasmodium-associated coinfections.
Acknowledgments
We thank Dr. Jose Stoute for providing the P. chabaudi and technical
support facilitating use of the parasite. We thank Nancy Fiore for technical
assistance with the project.
Disclosures
The authors have no financial conflicts of interest.
References
1. Utzinger, J., and D. de Savigny. 2006. Control of neglected tropical diseases:
integrated chemotherapy and beyond. PLoS Med. 3: e112.
2. Brooker, S., W. Akhwale, R. Pullan, B. Estambale, S. E. Clarke, R. W. Snow, and
P. J. Hotez. 2007. Epidemiology of plasmodium-helminth co-infection in Africa:
populations at risk, potential impact on anemia, and prospects for combining
control. Am. J. Trop. Med. Hyg. 77(Suppl. 6): 88–98.
3. Hartgers, F. C., and M. Yazdanbakhsh. 2006. Co-infection of helminths and
malaria: modulation of the immune responses to malaria. Parasite Immunol. 28:
497–506.
4. Haque, A., N. Rachinel, M. R. Quddus, S. Haque, L. H. Kasper, and
E. Usherwood. 2004. Co-infection of malaria and g-herpesvirus: exacerbated
lung inflammation or cross-protection depends on the stage of viral infection.
Clin. Exp. Immunol. 138: 396–404.
5. World Health Organization. 2011. World Malaria Report. World Health Organization, Geneva, Switzerland.
6. Abdullah, S., K. Adazu, H. Masanja, D. Diallo, A. Hodgson, E. Ilboudo-Sanogo,
A. Nhacolo, S. Owusu-Agyei, R. Thompson, T. Smith, and F. N. Binka. 2007.
Patterns of age-specific mortality in children in endemic areas of sub-Saharan
Africa. Am. J. Trop. Med. Hyg. 77(Suppl. 6): 99–105.
7. Maina, R. N., D. Walsh, C. Gaddy, G. Hongo, J. Waitumbi, L. Otieno,
D. Jones, and B. R. Ogutu. 2010. Impact of Plasmodium falciparum infection
on haematological parameters in children living in Western Kenya. Malar. J. 9
(Suppl. 3): S4.
8. Bryce, J., C. Boschi-Pinto, K. Shibuya, and R. E. Black, and WHO Child Health
Epidemiology Reference Group. 2005. WHO estimates of the causes of death in
children. Lancet 365: 1147–1152.
9. Patriarca, P. A., P. F. Wright, and T. J. John. 1991. Factors affecting the immunogenicity of oral poliovirus vaccine in developing countries: review. Rev.
Infect. Dis. 13: 926–939.
10. Tarzaali, A., P. Viens, and M. Quevillon. 1977. Inhibition of the immune response to whooping cough and tetanus vaccines by malaria infection, and the
effect of pertussis adjuvant. Am. J. Trop. Med. Hyg. 26: 520–524.
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
time or after the infection was resolved. Previous studies found
no effect on OVA-specific Ab responses when OVA was injected
before P. chabaudi infection (31) or when NP-CGG was given at
the same time as P. chabaudi infection (30). The effect of timing
of the secondary Ag delivery during P. chabaudi infection could
be the result of the immune system being primed early in malaria
infection toward a Th1 response or because of the presence of
a variety of parasite pathogen–associated molecular patterns that
are capable of stimulating B cells (30, 52).
Coinfections occurring alongside malaria can consist of many
different types of Ag and pathogens. The ability of acute P. chabaudi to decrease TD and increase TI secondary Ag-specific Ab
responses suggests that the nature of the Ag is critical during coinfection. Enhancement of TI Ab responses by P. chabaudi infection could be due in part to the cytokine milieu induced by acute
infection. There is evidence that IL-12 signals are capable of enhancing Ab responses to polysaccharide TI vaccines (53), and IL-12
expression is important to immunity to P. chabaudi (54). In addition, B cell priming via TLR signaling or other pattern recognition
receptors could lead to enhanced TI Ab responses (55). TI Ab enhancement could happen in addition to a productive TD Ab response
and not necessarily instead of a robust TD response. It is likely
that there are several different mechanisms in play during malaria
infection that lead to changes in Ab responses to secondary Ags and
pathogens.
The progression of B cells from naive cells to Ab-producing
cells involves a tightly regulated process dependent on migration
between niches within secondary lymphoid tissues. The kinetics
of GC responses to NP-coupled TD Ags (e.g., OVA or CGG) were
defined by Jacob et al. (24). Following treatment of mice with
NP-CGG, GCs containing B cells undergoing somatic hypermutation appear in the spleen 8–10 d later and persist until at least
16 d postimmunization. GCs induced as a consequence of parasitic infections, including P. chabaudi, demonstrate similar GC
kinetics (56, 57). However, it is clear that infection with Plasmodium affects the architecture of the spleen (30), which is a key
immune induction site for blood-borne pathogens. Carvalho et al.
(43) observed that GCs in the spleen lost their conventional
structure following infection with Plasmodium berghei and reported extrafollicular development of plasmablasts. In addition,
extrafollicular B cell activation has been reported for P. chabaudi
(57) and other parasite infections (58). Disruption of the GC reaction has been shown to result in diminished production of classswitched, high-affinity Abs (59). In addition, P. chabaudi infection
has been shown to affect B cell lymphopoiesis and the maturation
of B cells into marginal zone or follicular B cells (60). These
defects in B cell development could impact GC responses and the
development of both B cell memory and long-lived Ab responses.
Extrafollicular B cell activation in the spleen is characterized
by the generation of short-lived plasmablasts that secrete Abs
with lower affinity for Ag (61). In the current study, we demonstrated that P. chabaudi is capable of inducing an increase in plasma
cells at 17 dpi, and depletion of IFN-g resulted in significantly
increased plasma cell percentages in both NP-OVA control and
P. chabaudi– and NP-OVA–injected mice. At present, it is unclear
whether malaria modulates signals important to either the organization of the GC such as CXCL13 (62, 63) or lymphocyte migration signals involving sphingosine 1-phosphate (64) that are
critical to the GC reaction. The lack of detectable NP+ cells in
GCs following NP-OVA injection of P. chabaudi–infected mice,
but the presence of NP-specific Ab suggests that the response to
NP-OVA is extrafollicular during P. chabaudi infection. The enhancement of NP-specific Ab titers following TI Ag during P.
chabaudi infection also suggests a shift to extrafollicular B cell
7
8
MALARIA AFFECTS HUMORAL RESPONSES TO SECONDARY Ag
37. Kinyanjui, S. M., P. Bejon, F. H. Osier, P. C. Bull, and K. Marsh. 2009. What you
see is not what you get: implications of the brevity of antibody responses to
malaria antigens and transmission heterogeneity in longitudinal studies of malaria
immunity. Malar. J. 8: 242.
38. Kinyanjui, S. M., D. J. Conway, D. E. Lanar, and K. Marsh. 2007. IgG antibody
responses to Plasmodium falciparum merozoite antigens in Kenyan children
have a short half-life. Malar. J. 6: 82.
39. Früh, K., O. Doumbo, H. M. Müller, O. Koita, J. McBride, A. Crisanti, Y. Touré,
and H. Bujard. 1991. Human antibody response to the major merozoite surface
antigen of Plasmodium falciparum is strain specific and short-lived. Infect.
Immun. 59: 1319–1324.
40. Cavanagh, D. R., I. M. Elhassan, C. Roper, V. J. Robinson, H. Giha,
A. A. Holder, L. Hviid, T. G. Theander, D. E. Arnot, and J. S. McBride. 1998. A
longitudinal study of type-specific antibody responses to Plasmodium falciparum
merozoite surface protein-1 in an area of unstable malaria in Sudan. J. Immunol.
161: 347–359.
41. Weiss, G.E., B. Traore, K. Kayentao, A. Ongoiba, S. Doumbo, D. Doumtabe,
Y. Kone, S. Dia, A. Guindo, A. Traore, et al. 2010. The Plasmodium falciparum‑specific human memory B cell compartment expands gradually with repeated
malaria infections. PLoS Pathog. 6: e1000912.
42. Nduati, E. W., D. H. Ng, F. M. Ndungu, P. Gardner, B. C. Urban, and
J. Langhorne. 2010. Distinct kinetics of memory B-cell and plasma-cell
responses in peripheral blood following a blood-stage Plasmodium chabaudi
infection in mice. PLoS One 5: e15007.
43. Carvalho, L. J., M. F. Ferreira-da-Cruz, C. T. Daniel-Ribeiro, M. PelajoMachado, and H. L. Lenzi. 2007. Germinal center architecture disturbance
during Plasmodium berghei ANKA infection in CBA mice. Malar. J. 6: 59.
44. Wykes, M. N., Y. H. Zhou, X. Q. Liu, and M. F. Good. 2005. Plasmodium yoelii
can ablate vaccine-induced long-term protection in mice. J. Immunol. 175:
2510–2516.
45. Portugal, S., S. K. Pierce, and P. D. Crompton. 2013. Young lives lost as B cells
falter: what we are learning about antibody responses in malaria. J. Immunol.
190: 3039–3046.
46. Cunningham, A. F., F. Gaspal, K. Serre, E. Mohr, I. R. Henderson, A. ScottTucker, S. M. Kenny, M. Khan, K. M. Toellner, P. J. Lane, and I. C. MacLennan.
2007. Salmonella induces a switched antibody response without germinal centers
that impedes the extracellular spread of infection. J. Immunol. 178: 6200–6207.
47. Finkelman, F. D., I. M. Katona, T. R. Mosmann, and R. L. Coffman. 1988. IFN-g
regulates the isotypes of Ig secreted during in vivo humoral immune responses. J.
Immunol. 140: 1022–1027.
48. Swanson, C. L., T. J. Wilson, P. Strauch, M. Colonna, R. Pelanda, and
R. M. Torres. 2010. Type I IFN enhances follicular B cell contribution to the
T cell‑independent antibody response. J. Exp. Med. 207: 1485–1500.
49. Sharma, S., R. B. DeOliveira, P. Kalantari, P. Parroche, N. Goutagny, Z. Jiang,
J. Chan, D. C. Bartholomeu, F. Lauw, J. P. Hall, et al. 2011. Innate immune
recognition of an AT-rich stem-loop DNA motif in the Plasmodium falciparum
genome. Immunity 35: 194–207.
50. de Goër de Herve, M. G., D. Durali, B. Dembele, M. Giuliani, T. A. Tran,
B. Azzarone, P. Eid, M. Tardieu, J. F. Delfraissy, and Y. Taoufik. 2011. Interferon-a triggers B cell effector 1 (Be1) commitment. PLoS One 6: e19366.
51. Harris, D. P., S. Goodrich, A. J. Gerth, S. L. Peng, and F. E. Lund. 2005.
Regulation of IFN-g production by B effector 1 cells: essential roles for T-bet
and the IFN-g receptor. J. Immunol. 174: 6781–6790.
52. Dostert, C., G. Guarda, J. F. Romero, P. Menu, O. Gross, A. Tardivel, M. L. Suva,
J. C. Stehle, M. Kopf, I. Stamenkovic, et al. 2009. Malarial hemozoin is a Nalp3
inflammasome activating danger signal. PLoS One 4: e6510.
53. Buchanan, R. M., B. P. Arulanandam, and D. W. Metzger. 1998. IL-12 enhances
antibody responses to T-independent polysaccharide vaccines in the absence of
T and NK cells. J. Immunol. 161: 5525–5533.
54. Stevenson, M. M., M. F. Tam, S. F. Wolf, and A. Sher. 1995. IL-12‑induced protection against blood-stage Plasmodium chabaudi AS requires IFN-g and TNF-a
and occurs via a nitric oxide-dependent mechanism. J. Immunol. 155: 2545–2556.
55. Snapper, C. M., and J. J. Mond. 1996. A model for induction of T cellindependent humoral immunity in response to polysaccharide antigens. J.
Immunol. 157: 2229–2233.
56. Amezcua Vesely, M. C., D. A. Bermejo, C. L. Montes, E. V. Acosta-Rodrı́guez,
and A. Gruppi. 2012. B-cell response during protozoan parasite infections. J.
Parasitol. Res. 2012: 362131.
57. Achtman, A. H., M. Khan, I. C. MacLennan, and J. Langhorne. 2003. Plasmodium chabaudi chabaudi infection in mice induces strong B cell responses and
striking but temporary changes in splenic cell distribution. J. Immunol. 171: 317–324.
58. Bermejo, D. A., M. C. Amezcua Vesely, M. Khan, E. V. Acosta Rodrı́guez,
C. L. Montes, M. C. Merino, K. M. Toellner, E. Mohr, D. Taylor,
A. F. Cunningham, and A. Gruppi. 2011. Trypanosoma cruzi infection induces
a massive extrafollicular and follicular splenic B-cell response which is a high
source of non‑parasite-specific antibodies. Immunology 132: 123–133.
59. Han, S., X. Zhang, G. Wang, H. Guan, G. Garcia, P. Li, L. Feng, and B. Zheng.
2004. FTY720 suppresses humoral immunity by inhibiting germinal center reaction. Blood 104: 4129–4133.
60. Bockstal, V., N. Geurts, and S. Magez. 2011. Acute disruption of bone marrow
B lymphopoiesis and apoptosis of transitional and marginal zone B cells in
the spleen following a blood-stage Plasmodium chabaudi infection in mice. J.
Parasitol. Res. 2011: 534697.
61. MacLennan, I. C., K. M. Toellner, A. F. Cunningham, K. Serre, D. M. Sze,
E. Zúñiga, M. C. Cook, and C. G. Vinuesa. 2003. Extrafollicular antibody
responses. Immunol. Rev. 194: 8–18.
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
11. Usen, S., P. Milligan, C. Ethevenaux, B. Greenwood, and K. Mulholland. 2000.
Effect of fever on the serum antibody response of Gambian children to Haemophilus influenzae type b conjugate vaccine. Pediatr. Infect. Dis. J. 19: 444–
449.
12. Williamson, W. A., and B. M. Greenwood. 1978. Impairment of the immune
response to vaccination after acute malaria. Lancet 1: 1328–1329.
13. Grau, G. E., T. E. Taylor, M. E. Molyneux, J. J. Wirima, P. Vassalli, M. Hommel,
and P. H. Lambert. 1989. Tumor necrosis factor and disease severity in children
with falciparum malaria. N. Engl. J. Med. 320: 1586–1591.
14. Shear, H. L., R. Srinivasan, T. Nolan, and C. Ng. 1989. Role of IFN-g in lethal
and nonlethal malaria in susceptible and resistant murine hosts. J. Immunol. 143:
2038–2044.
15. Langhorne, J., S. Gillard, B. Simon, S. Slade, and K. Eichmann. 1989. Frequencies of CD4+ T cells reactive with Plasmodium chabaudi chabaudi: distinct
response kinetics for cells with Th1 and Th2 characteristics during infection. Int.
Immunol. 1: 416–424.
16. Taylor-Robinson, A. W., and R. S. Phillips. 1994. B cells are required for the
switch from Th1- to Th2-regulated immune responses to Plasmodium chabaudi
chabaudi infection. Infect. Immun. 62: 2490–2498.
17. Yuan, R. R., G. Spira, J. Oh, M. Paizi, A. Casadevall, and M. D. Scharff. 1998.
Isotype switching increases efficacy of antibody protection against Cryptococcus
neoformans infection in mice. Infect. Immun. 66: 1057–1062.
18. Coutelier, J. P., J. T. van der Logt, F. W. Heessen, A. Vink, and J. van Snick.
1988. Virally induced modulation of murine IgG antibody subclasses. J. Exp.
Med. 168: 2373–2378.
19. Taylor, R. R., D. B. Smith, V. J. Robinson, J. S. McBride, and E. M. Riley. 1995.
Human antibody response to Plasmodium falciparum merozoite surface protein
2 is serogroup specific and predominantly of the immunoglobulin G3 subclass.
Infect. Immun. 63: 4382–4388.
20. Garraud, O., R. Perraut, A. Diouf, W. S. Nambei, A. Tall, A. Spiegel,
S. Longacre, D. C. Kaslow, H. Jouin, D. Mattei, et al. 2002. Regulation of
antigen-specific immunoglobulin G subclasses in response to conserved and
polymorphic Plasmodium falciparum antigens in an in vitro model. Infect.
Immun. 70: 2820–2827.
21. Tongren, J. E., P. H. Corran, W. Jarra, J. Langhorne, and E. M. Riley. 2005.
Epitope-specific regulation of immunoglobulin class switching in mice immunized with malarial merozoite surface proteins. Infect. Immun. 73: 8119–8129.
22. Ndungu, F. M., E. T. Cadman, J. Coulcher, E. Nduati, E. Couper, D. W. Macdonald,
D. Ng, and J. Langhorne. 2009. Functional memory B cells and long-lived plasma
cells are generated after a single Plasmodium chabaudi infection in mice. PLoS
Pathog. 5: e1000690.
23. Barr, T. A., S. Brown, P. Mastroeni, and D. Gray. 2009. B cell intrinsic MyD88
signals drive IFN-g production from T cells and control switching to IgG2c. J.
Immunol. 183: 1005–1012.
24. Jacob, J., R. Kassir, and G. Kelsoe. 1991. In situ studies of the primary immune
response to (4-hydroxy-3-nitrophenyl)acetyl. I. The architecture and dynamics of
responding cell populations. J. Exp. Med. 173: 1165–1175.
25. Ferrante, A., L. J. Beard, and R. G. Feldman. 1990. IgG subclass distribution of
antibodies to bacterial and viral antigens. Pediatr. Infect. Dis. J. 9(Suppl. 8):
S16–S24.
26. Lalor, P. A., G. J. Nossal, R. D. Sanderson, and M. G. McHeyzer-Williams.
1992. Functional and molecular characterization of single, (4-hydroxy-3nitrophenyl)acetyl (NP)-specific, IgG1 + B cells from antibody-secreting and
memory B cell pathways in the C57BL/6 immune response to NP. Eur. J.
Immunol. 22: 3001–3011.
27. Mosmann, T. R., H. Cherwinski, M. W. Bond, M. A. Giedlin, and R. L. Coffman.
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.
28. Mountford, A. P., A. Fisher, and R. A. Wilson. 1994. The profile of IgG1 and
IgG2a antibody responses in mice exposed to Schistosoma mansoni. Parasite
Immunol. 16: 521–527.
29. Stephens, R., R. L. Culleton, and T. J. Lamb. 2012. The contribution of
Plasmodium chabaudi to our understanding of malaria. Trends Parasitol. 28:
73–82.
30. Cadman, E. T., A. Y. Abdallah, C. Voisine, A. M. Sponaas, P. Corran, T. Lamb,
D. Brown, F. Ndungu, and J. Langhorne. 2008. Alterations of splenic architecture in malaria are induced independently of Toll-like receptors 2, 4, and 9 or
MyD88 and may affect antibody affinity. Infect. Immun. 76: 3924–3931.
31. Sardinha, L. R., M. R. D’Império Lima, and J. M. Alvarez. 2002. Influence
of the polyclonal activation induced by Plasmodium chabaudi on ongoing OVAspecific B- and T-cell responses. Scand. J. Immunol. 56: 408–416.
32. Eaton, S. M., E. M. Burns, K. Kusser, T. D. Randall, and L. Haynes. 2004. Agerelated defects in CD4 T cell cognate helper function lead to reductions in humoral responses. J. Exp. Med. 200: 1613–1622.
33. Maizels, N., J. C. Lau, P. R. Blier, and A. Bothwell. 1988. The T-cell independent antigen, NP-Ficoll, primes for a high affinity IgM anti-NP response.
Mol. Immunol. 25: 1277–1282.
34. Garcı́a de Vinuesa, C., P. O’Leary, D. M. Sze, K. M. Toellner, and
I. C. MacLennan. 1999. T-independent type 2 antigens induce B cell proliferation in multiple splenic sites, but exponential growth is confined to extrafollicular foci. Eur. J. Immunol. 29: 1314–1323.
35. Mosmann, T. R., and R. L. Coffman. 1989. TH1 and TH2 cells: different patterns
of lymphokine secretion lead to different functional properties. Annu. Rev.
Immunol. 7: 145–173.
36. Cohen, S., I. A. McGregor, and S. Carrington. 1961. Gamma-globulin and acquired immunity to human malaria. Nature 192: 733–737.
The Journal of Immunology
62. Cyster, J. G. 2010. B cell follicles and antigen encounters of the third kind. Nat.
Immunol. 11: 989–996.
63. Allen, C. D., K. M. Ansel, C. Low, R. Lesley, H. Tamamura, N. Fujii, and
J. G. Cyster. 2004. Germinal center dark and light zone organization is mediated
by CXCR4 and CXCR5. Nat. Immunol. 5: 943–952.
64. Green, J. A., K. Suzuki, B. Cho, L. D. Willison, D. Palmer, C. D. Allen,
T. H. Schmidt, Y. Xu, R. L. Proia, S. R. Coughlin, and J. G. Cyster. 2011. The
sphingosine 1-phosphate receptor S1P₂ maintains the homeostasis of germinal center B cells and promotes niche confinement. Nat. Immunol. 12:
672–680.
9
65. Sponaas, A. M., N. Belyaev, M. Falck-Hansen, A. Potocnik, and J. Langhorne.
2012. Transient deficiency of dendritic cells results in lack of a merozoite surface
protein 1-specific CD4 T cell response during peak Plasmodium chabaudi bloodstage infection. Infect. Immun. 80: 4248–4256.
66. Perry, J. A., A. Rush, R. J. Wilson, C. S. Olver, and A. C. Avery. 2004. Dendritic cells
from malaria-infected mice are fully functional APC. J. Immunol. 172: 475–482.
67. Illingworth, J., N. S. Butler, S. Roetynck, J. Mwacharo, S. K. Pierce, P. Bejon,
P. D. Crompton, K. Marsh, and F. M. Ndungu. 2013. Chronic exposure to
Plasmodium falciparum is associated with phenotypic evidence of B and T cell
exhaustion. J. Immunol. 190: 1038–1047.
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017