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INFECTION AND IMMUNITY, May 2009, p. 1924–1935 Vol. 77, No.
50019-9567/09/$08.00�0 doi:10.1128/IAI.01559-08Copyright © 2009,
American Society for Microbiology. All Rights Reserved.
Cholera Toxin and Escherichia coli Heat-Labile Enterotoxin, but
NotTheir Nontoxic Counterparts, Improve the Antigen-Presenting
Cell
Function of Human B Lymphocytes�
Donatella R. M. Negri,1* Dora Pinto,1 Silvia Vendetti,1 Mario
Patrizio,2 Massimo Sanchez,3Antonella Riccomi,1 Paolo Ruggiero,4
Giuseppe Del Giudice,4
and Maria Teresa De Magistris1
Department of Infectious, Parasitic and Immune-Mediated
Diseases,1 Department of Therapeutic Research andMedicines
Evaluation,2 and Department of Cell Biology and Neurosciences,3
Istituto Superiore di Sanità,
Rome, Italy, and Research Center, Novartis Vaccines, Siena,
Italy4
Received 23 December 2008/Accepted 9 February 2009
B lymphocytes play an important role in the immune response
induced by mucosal adjuvants. In this studywe investigated the in
vitro antigen-presenting cell (APC) properties of human B cells
upon treatment withcholera toxin (CT) and Escherichia coli
heat-labile enterotoxin (LT) and nontoxic counterparts of these
toxins,such as the B subunit of CT (CT-B) and the mutant of LT
lacking ADP ribosyltransferase activity (LTK63).Furthermore,
forskolin (FSK), a direct activator of adenylate cyclase, and
cyclic AMP (cAMP) analogues wereused to investigate the role of the
increase in intracellular cAMP caused by the A subunit of CT and
LT. Blymphocytes were cultured with adjuvants and polyclonal
stimuli necessary for activation of B cells in theabsence of CD4 T
cells. Data indicated that treatment with CT, LT, FSK, or cAMP
analogues, but not treatmentwith CT-B or LTK63, upregulated surface
activation markers on B cells, such as CD86 and HLA-DR, andinduced
inhibition of the proliferation of B cells at early time points,
while it increased cell death in long-termcultures. Importantly, B
cells treated with CT, LT, or FSK were able to induce pronounced
proliferation of bothCD4� and CD8� allogeneic T cells compared with
untreated B cells and B cells treated with CT-B and LTK63.Finally,
only treatment with toxins or FSK induced antigen-specific T-cell
proliferation in Mycobacteriumtuberculosis purified protein
derivative or tetanus toxoid responder donors. Taken together,
these resultsindicated that the in vitro effects of CT and LT on
human B cells are mediated by cAMP.
The development of effective mucosal vaccines has beenhindered
by the lack of useful adjuvants and our limited knowl-edge of their
modes of action. Cholera toxin (CT) from Vibriocholerae and
Escherichia coli heat-labile enterotoxin (LT) arepotent
immunological adjuvants, as indicated by mouse vac-cine studies,
although their mechanisms of action are not fullyunderstood. These
toxins are holotoxins composed of an en-zymatically active A
subunit that is noncovalently linked to apentamer of B subunits
binding a variety of galactose-contain-ing molecules present in the
plasma membranes of eukaryoticcells. CT binds mostly to the
ganglioside GM1, which is be-lieved to be the major toxin receptor,
whereas LT binds notonly to GM1 but also to other
glycosphingolipids. Once inter-nalized, the A subunit ADP
ribosylates the � subunit of theGTP-binding regulatory protein Gs,
thereby inducing perma-nent adenylate cyclase activation, resulting
in an increase in thelevel of intracellular cyclic AMP (cAMP)
(reviewed in refer-ence 34).
The potentiation of antigen-presenting cell (APC) functionis a
major aspect of adjuvant action, and it has been shown thatCT and
LT induce maturation of both murine dendritic cells(DC) (26, 36)
and human DC (5, 14, 15). Several studies
demonstrated the ability of these toxins to promote
B-cellisotype switch differentiation in mice (19, 27) and
upregulationof activation markers in both murine and human B cells
(2–4).While these toxins are potent adjuvants, their toxicity
makesthem unsuitable for human use. For this reason, a number
ofinvestigators have tried to develop nontoxic derivatives of CTand
LT that retain adjuvanticity either by removing the Adomain or by
rendering it enzymatically inactive by site-di-rected mutagenesis
(34). Although the current data suggestthat the enzymatic activity
of CT and LT holotoxins is respon-sible for the most potent
adjuvant activity, a number of reportsproposed that there are
multiple immune modulating pathwaysthat are triggered by CT and LT,
including mechanisms inde-pendent of ADP ribosyltransferase
activity (11, 13, 30, 33, 42).Numerous studies have suggested that
engagement of the gan-glioside GM1, the major receptor for CT and
LT, is requiredfor the ability of these molecules to modulate
immune re-sponses (22, 31). Recently, workers demonstrated that in
theabsence of the toxic A subunit, the B subunit of CT
(CT-B)induces intracellular signaling associated with the in vitro
ac-tivation of murine B cells and macrophages (37).
The majority of these studies have been performed withmurine
cells and have confirmed the in vivo adjuvanticity ofnontoxic
compounds, such as CT-B and LTK63, a mutant ofLT lacking the ADP
ribosyltransferase enzymatic activity,when they were mucosally
delivered into animals, even if theimmune responses observed in the
in vivo studies were usuallyweaker than those induced by the
wild-type toxins (6, 11, 20,
* Corresponding author. Mailing address: Department of
Infectious,Parasitic and Immune-Mediated Diseases, Istituto
Superiore di Sanità,Viale Regina Elena 299, 00161 Rome, Italy.
Phone: 39-6-49902734.Fax: 39-6-49902886. E-mail:
[email protected].
� Published ahead of print on 17 February 2009.
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36, 40, 41). In order to develop a mucosal adjuvant for
humanvaccine, the mechanism(s) of action of potential nontoxic
ad-juvants should be investigated in vitro by using human APC.
Ithas been shown that the B-cell antigen-presenting functionsmay be
important for the induction of optimal vaccine-inducedresponses
(10, 35). Moreover, B cells are present in mucosa-associated
lymphoid tissues (8), and their function in thesesites is related
not only to immunoglobulin (Ig) production butalso to their
antigen-presenting properties (24). To elucidatethe mechanisms by
which enterotoxins modulate antigen-pre-senting properties, we
decided to carry out a comprehensiveand comparative analysis of the
effects of the toxins and theirnontoxic derivatives on the APC
function of human B cells.Here we present evidence that CT and LT,
as well as forskolin(FSK) and cAMP analogues, but not CT-B and
LTK63, in-crease the activation of human B cells and induce
improve-ment in their APC capability, indicating that the presence
ofthe enzymatic subunit is critical for their adjuvanticity.
MATERIALS AND METHODS
Recombinant enterotoxins. CT and CT-B were purchased from List
BiologicalLaboratories (Campbell, CA); E. coli LT was purchased
from Swiss SerumVaccine Institute (Berne, Switzerland); LTK63 was
provided by Novartis (Siena,Italy); and FSK, dibutyryl-cAMP
(Db-cAMP), and 8-bromo-cAMP (8Br-cAMP)were purchased from Sigma
Chemical Co. (St. Louis, MO). Endotoxin contam-ination in adjuvant
preparations was evaluated by Limulus amoebocyte lysateanalysis
(Pyrochrome; Associates of Cape Cod, Falmouth, MA). The
concentra-tion of endotoxin was less than 0.09 endotoxin unit /�g
in all of the preparationsutilized in this study.
Isolation and activation of B cells. Human B cells were isolated
from periph-eral blood mononuclear cells (PBMC) from healthy donors
by positive selectionusing anti-CD19 microbeads and the
manufacturer’s suggested protocol (Milte-nyi Biotec S.r.l.,
Bologna, Italy). The cells obtained were �95% CD19 positive,as
assessed by flow cytometry analysis. B cells were cultured in
24-well plates orin 96-well plates at a concentration of 1.5 � 106
to 2 � 106 cells/ml in RPMI 1640medium (GIBCO Invitrogen, Paisley,
United Kingdom) supplemented with 100U/ml of
penicillin-streptomycin-glutamine (GIBCO Invitrogen, Paisley,
UnitedKingdom), 10% heat-inactivated fetal bovine serum (Euroclone,
Life SciencesDivision, Pero, Italy), sodium pyruvate (Euroclone),
and nonessential aminoacids (Euroclone). In order to obtain
polyclonal stimulation, B cells were cul-tured in the presence of
2.5 �g/ml of CpG ODN 2006 (MWG Biotech, M-Medical, Milan, Italy),
50 U/ml of interleukin-2 (IL-2) (BD Biosciences, SanDiego, CA), and
2 �g/ml of anti-Ig monoclonal antibody (MAb) (Jackson
Im-munoResearch Laboratories, Suffolk, United Kingdom). In
addition, togetherwith the stimuli, on day zero B cells were either
treated with 3 �g/ml of CT, 10�g/ml of CT-B, 0.1 �g/ml of LT, 10
�g/ml of LTK63, 50 �M of FSK, 0.5 mMDb-cAMP, or 8Br-cAMP or left
untreated. In some experiments CD27� andCD27� B cells were isolated
by sorting total B cells with FACSAria (BD Bio-sciences, San Diego,
CA). Briefly, B cells were isolated by using anti-CD19microbeads,
as described above. CD27� and CD27� B-cell subsets were
purifiedbased on CD27 cell surface expression by FACSAria after
staining with phyco-erythrin-conjugated anti-CD27 MAb
(Immunological Sciences, Rome, Italy).Dead cells were excluded on
the basis of propidium iodide (PI) (5 �g/ml; BDBiosciences)
fluorescence intensity. The two subpopulations were stimulatedwith
polyclonal stimuli and treated with adjuvants as described above
for theunfractionated B cells.
Determination of intracellular cAMP content. B cells (2 � 105
cells in 200 �l,seeded in duplicate) were stimulated and treated
for 24 h with adjuvants or FSKor left untreated in the presence of
100 �M 3-isobutyl-1-methylxanthine (SigmaChemical Co., St. Louis,
MO), which inhibits cAMP-hydrolyzing phosphodies-terases, in order
to avoid cAMP degradation. The culture medium was removedafter 10
min of centrifugation at 1,300 rpm, and cold 0.1 N HCl was used to
lysethe cells. The intracellular cAMP content was measured by an
enzyme-linkedimmunoassay by following the manufacturer’s
instructions (Biotrak EIA, GEHealthcare).
Flow cytometric immunofluorescence analysis of surface markers
and apop-totic cells. B cells were stained with the following mouse
anti-human MAbsobtained from Becton Dickinson (BD Biosciences, San
Diego, CA): phyco-
erythrin-labeled anti-CD86, anti-HLA class I, anti-CD80, and
anti-CD40 andperidinin chlorophyll protein-labeled anti-CD20 and
anti-HLA class II. Isotype-matched mouse IgG MAbs were used as
controls. To evaluate B-cell death,stimulated B cells, treated as
indicated above for 3 and 5 days, were stained withAnnexin
V-fluorescein isothiocyanate (FITC) plus PI (Annexin V-FITC
apop-tosis detection kit II; BD PharMingen, San Diego, CA) by
following the manu-facturer’s instructions. Flow cytometric
analysis of the cells was performed usinga FACSCalibur and
CellQuest software (BD Biosciences).
B-cell proliferation assay. PBMC or purified B cells were
labeled with 2.5 �Mcarboxyfluorescein succinimidyl ester (CFSE)
(Molecular Probes, Eugene, OR)in phosphate-buffered saline
containing 1% fetal bovine serum for 10 min at37°C, washed in
complete RPMI 1640 medium, and then seeded in 24-well or96-well
culture plates (1.5 � 106 cells/ml) containing polyclonal stimuli
(seeabove) and subjected to different treatments. After 3 and 5
days of culture, thecells were washed and stained with MAbs against
human CD4, CD8, or CD20from Becton Dickinson. The amounts of cell
proliferation in the cell populationswere quantified by monitoring
the sequential loss of fluorescence intensity of theCFSE-labeled
cells using a FACSCalibur.
Mixed allogeneic cultures and antigen-specific presentation
assay. Purified,stimulated B cells treated as indicated above or
left untreated for 3 days, care-fully washed, and irradiated (3,000
rads) were cocultured with allogeneic PBMClabeled with CFSE (see
above). A total of 1 � 105 PBMC per well were seededonto 96-well
plates (Sigma-Aldrich S.r.l., Milan, Italy) with titrated numbers
ofirradiated B cells (the B cell/PBMC ratio ranged from 1:16 to
1:1). After 3 daysof coculture, cells were collected and stained
with anti-human CD4 or anti-human CD8 MAbs. The levels of PBMC,
CD4�, and CD8� T-cell proliferationwere evaluated by
fluorescence-activated cell sorting (FACS) analysis. To exam-ine
the antigen-specific response, B cells isolated from Mycobacterium
tubercu-losis purified protein derivative (PPD) responder donors or
tetanus toxoid (TT)responder donors were stimulated with CpG, IL-2,
and PPD (Statens SerumInstitute, Copenhagen, Denmark) or with TT
(Novartis, Siena, Italy) at day zero.Toxins, CT-B, LTK63, and FSK
were added on the same day. PPD and TT werealso added on day 2. On
day 3, cells were extensively washed, irradiated, andcocultured
with autologous PBMC previously labeled with CFSE. CD4� andCD8�
T-cell proliferation was evaluated after 5 days of coculture using
a FAC-SCalibur and CellQuest software.
In some experiments anti-human CD86 and/or anti-human HLA-DR
MAbswere used in order to block the interaction between APC and T
cells in a mixedleukocyte reaction (MLR) assay. Briefly, B cells
were incubated for 2 h at 4°Cwith 20 �g/ml of anti-CD86 (BU63;
mouse IgG1; Ancell Immunology ResearchProducts, Bayport, MN) and/or
anti-HLA-DR (G46-6; mouse IgG2a; BD Bio-sciences, San Diego, CA)
MAbs, extensively washed to remove free MAbs, andthen added to
CFSE-labeled PBMC.
Cytokine production. Cytokine concentrations in supernatants
collected fromstimulated B cells treated as indicated above or left
untreated for 3 days weredetermined by enzyme-linked immunosorbent
assays (ELISA), including assaysfor tumor necrosis factor alpha
(TNF-�) and IL-1� (Pierce Endogen, Rockford,IL), IL-6 (BD
Biosciences, San Diego, CA), and IL-12 (R&D Systems,
Minne-apolis, MN).
Statistical analysis. Microsoft Excel (Microsoft Corporation,
Redmond, WA)was used for statistical analysis. Data were expressed
as means � standarddeviations, and statistical significance was
determined by Student’s t test. A Pvalue of 0.05 was considered
statistically significant.
RESULTS
CT and LT induce upregulation of surface activation mark-ers.
The levels of expression of activation markers CD86,CD80, HLA class
I and II molecules, and CD40 on the B-cellsurface were examined
after treatment with CT, CT-B, LT,LTK63, FSK, or cAMP analogues,
such as Db-cAMP and8Br-cAMP, for 3 and 5 days. As expected,
stimulation of Bcells cultured in the presence of polyclonal
stimuli (stimulatedB cells), such as CpG ODN 2006, anti-Ig MAb, and
IL-2,induced upregulation of CD86 and HLA-DR markers that
wasdetectable after 3 days of culture compared to the results
forunstimulated B cells (Fig. 1). Treatment of both unstimulatedand
stimulated B cells with CT, LT, FSK, or cAMP analoguesinduced
upregulation of these markers. As shown in Fig. 1, for
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CD86 expression both the percentage of positive cells and
themean fluorescence intensity (MFI) were strongly
increased,whereas for HLA-DR, already expressed on the majority of
Bcells, there was a marked increase in MFI following treatmentwith
CT, LT, FSK, or cAMP analogues. None of the treat-ments was able to
induce an evident change in CD40 and HLAclass I expression, whereas
slight downregulation of CD80 ex-pression was observed in
stimulated B cells treated with toxin,FSK, or cAMP analogues (data
not shown). The presence ofCT-B or LTK63 in the culture did not
induce modulation ofthe expression of any of the markers analyzed
(Fig. 1). Asshown in dot plots in Fig. 1, isolated B cells cultured
withoutpolyclonal stimulation showed high mortality. For this
reasonwe decided to perform the next experiments in the presence
ofpolyclonal stimuli, which were also necessary for analysis
ofB-cell proliferation and cytokine production.
CT and LT induce an increase in the intracellular cAMPlevels. In
order to check the enzymatic activity of adjuvants,intracellular
cAMP was evaluated in B cells treated with thedifferent compounds.
As expected, CT, LT, and FSK, but notCT-B or LTK63, induced
increases in intracellular cAMP lev-els in B cells (Fig. 2). The
results showed that there was astatistically significant difference
(P 0.05) between B cellstreated with CT, LT, or FSK and untreated
control (5,436 �1,001, 5,054 � 1,142, 11,613 � 2,178, and 1,458 �
266 fmol/106
cells, respectively). Moreover, FSK-treated B cells
containedstatistically significant larger amounts of intracellular
cAMPthan toxin-treated cells (Fig. 2).
CT and LT inhibit proliferation of B cells and increase
theirsusceptibility to death. In order to evaluate the effect of
adju-vants on cell proliferation, B lymphocytes were stained
withCSFE and cultured for 3 and 5 days with polyclonal stimuli
andwith CT, CT-B, LT, LTK63, FSK, or cAMP analogues or
leftuntreated. Figure 3 shows the results of a representative
ex-periment. Polyclonal stimuli induced proliferation after 3
daysof culture (67.7% proliferating B cells), which increased
after5 days (83.1%). Similar percentages of dividing B cells
wereevident when the cells were treated with CT-B or LTK63(65.8%
and 63.6%, respectively, at 3 days and 81.7% and
86.2%,respectively, at 5 days), suggesting that these compounds
didnot act on the ability of stimulated B cells to proliferate.
Con-versely, proliferation of CT-treated B cells, and to a
lesserextent LT-treated B cells, was significantly less (P 0.05)
bothat day 3 (35.3% and 45.5%, respectively) and at day 5 (62.9%and
71.7%, respectively). Similar results were obtained when Bcells
were treated with Db-cAMP and 8Br-cAMP (37.1% and37.5%,
respectively, at day 3 and 57.7% and 57.1%, respec-tively, at day
5). The inhibition of cell proliferation induced bytoxins was
evident not only based on the percentage of prolif-erating cells
but also based on MFI, which indirectly indicatedthe rounds of
B-cell division under each condition analyzed(Fig. 3). When
stimulated B cells were treated with FSK, theirability to
proliferate was inhibited even more than it was whenthey were
treated with toxins (18.7% at day 3 and 25.3% at day5), probably
because of the high dose of FSK used (50 �M).The selection of this
dose was based on the observation thatlower doses were not
sufficient to induce activation of humanDC (5).
At the same time, in order to understand if the
evidentinhibition of proliferation observed was related to an
increasein B-cell death, the effects of toxins on B-cell viability
wereevaluated. The percentage of live cells was initially assessed
bygating the events (R1) on a dot plot with forward and sidescatter
parameters (Fig. 4A). On day 3, even if treatment withtoxins, FSK,
and cAMP analogues resulted in a slight reductionin viability, the
viability of B cells was high under all conditionstested,
suggesting that at this time cell death was not the causeof the
marked inhibition of B-cell proliferation described above.After 5
days of culture the percentage of live B cells decreased tothe same
level in the control (untreated) or CT-B- or LTK63-treated samples.
Treatment with FSK resulted in a further de-crease in viability (P
0.05), whereas CT, LT, and cAMP ana-logues induced a remarkable
reduction in viability (P 0.05). Toconfirm these results, we
labeled the cells with Annexin V and PIafter 3 and 5 days of
culture in the presence or absence of adju-vants or FSK. As shown
by the results of a representative exper-iment (Fig. 4B), after 3
days of culture the percentages of labeledcells (PI-positive cells,
Annexin V-positive cells, and PI- and An-
FIG. 1. Effect of adjuvants on the expression of cell surface
activation markers. Enzymatic activity is required for CT and LT to
upregulateactivation markers on the B-cell surface. Unstimulated B
cells (left panel) or B cells stimulated with 2.5 �g/ml of CpG ODN
2006, 50 U/ml of IL-2,and 2 �g/ml of anti-Ig MAb (right panel) were
simultaneously treated with the compounds indicated or left
untreated (NT) for 3 days. Theexpression of cell surface markers
was evaluated by FACS analysis of B cells stained with MAbs
directed to CD86 and HLA-DR. Dot plots withforward scatter (FSC)
and side scatter (SSC) parameters are shown to visualize the
amounts of live cells (in the gate) analyzed for surface
markers.The percentage of positive cells and the MFI are indicated
in each graph. Representative data from five independent
experiments are shown.PerCP, peridinin chlorophyll protein.
FIG. 2. Effect of adjuvants on intracellular cAMP. The level
ofintracellular cAMP increases upon treatment with CT, LT, or
FSK.Stimulated B cells were treated with the compounds indicated
for 24 hin the presence of 3-isobutyl-1-methylxanthine and lysed
with HCl.The amount of cAMP was evaluated by an enzyme immunoassay
andwas expressed in fmol/106 B cells. The asterisks indicate
statisticallysignificant differences (P 0.05) between CT-, LT-, or
FSK-treated Bcells and untreated B cells (NT). The P values
indicate the statisticallysignificant differences between FSK- and
CT-treated cells and betweenFSK- and LT-treated cells.
Representative data from three indepen-dent experiments are
shown.
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nexin V-positive cells) were similar under all conditions
analyzedexcept for LT-treated B cells, for which there was a slight
higherpercentage of total labeled cells. After 5 days, treatment
with CT,LT, or FSK resulted in evident increases in the percentages
of
both PI- and Annexin V-positive cells, suggesting that these
treat-ments induced increased susceptibility to death compared to
thatof B cells that were treated with CT-B or LTK63 or were
leftuntreated.
FIG. 3. Effect of adjuvants on B-cell proliferation. The
inhibitory effect of adjuvants on B-cell proliferation is related
to the increase in theintracellular cAMP level. CFSE-labeled B
cells were stimulated with polyclonal stimuli, including 2.5 �g/ml
of CpG ODN 2006, 50 U/ml of IL-2,and 2 �g/ml of anti-Ig MAb, and
simultaneously treated with the compounds indicated or left
untreated (NT) for 3 and 5 days. The percentagesof dividing cells
and the MFI of CFSE proliferating cells are indicated in the
graphs. The data shown are data from one representative
experimentof five experiments performed. DB, Db-cAMP; 8Br,
8Br-cAMP.
FIG. 4. Effect of adjuvants on B-cell viability. The enzymatic
activity of CT and LT renders B cells more susceptible to death in
long-termculture. B cells were stimulated with polyclonal stimuli,
including 2.5 �g/ml of CpG ODN 2006, 50 U/ml of IL-2, and 2 �g/ml
of anti-Ig MAb, andsimultaneously treated with the compounds
indicated or left untreated (NT) for 3 and 5 days. (A) Percentage
of live cells as evaluated by gatingthe events (R1) on a dot plot
with forward scatter (FSC) and side scatter (SSC) parameters. The
histogram shows the percentages of gated B cellsfrom five different
donors analyzed at day 3 (open bars) and day 5 (filled bars). The
error bars indicate standard deviations. Db, Db-cAMP; 8Br,8Br-cAMP.
(B) Stimulated B cells, treated as indicated for 3 and 5 days, were
stained with Annexin V-FITC plus PI. The percentages of
AnnexinV-positive cells, PI-positive cells, and Annexin V-positive
PI-positive cells are indicated in the plots. The data shown are
data from onerepresentative experiment of five experiments
performed.
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Toxin-treated B cells are efficient APC. In order to
investi-gate the APC function of stimulated B cells treated with
toxins,MLR assays were performed. In this setting, to maintain
agood activation state and to avoid cell death, B cells were usedas
APC after 3 days of stimulation and treatment. AllogeneicT-cell
proliferation was analyzed by FACS to determine thecontent of CFSE
after 3 days of coculture with irradiated Bcells as stimulators.
Figure 5 shows the proliferation of PBMCand CD4� and CD8� T cells
from three different donors. Bcells stimulated with polyclonal
stimuli were able to induce lowlevels of PBMC proliferation.
Treatment with toxins or withFSK clearly improved the APC function
of stimulated B cells,as shown by the increased proliferation of
allogeneic PBMC. Inparticular, CT-, LT-, or FSK-treated B cells
induced bothCD4� and CD8� T cells to proliferate at any ratio of B
cells toPBMC used (1:16 to 1:1) in a dose-dependent manner.
Theincrease in T-cell proliferation ranged from two- to
fivefoldcompared with the T-cell proliferation observed for the
un-treated control. Similar results were obtained when B cellswere
treated with Db-cAMP (data not shown). Conversely,treatment of B
cells with CT-B and LTK63 did not inducevariation in T-cell
proliferation compared to the results ob-tained with untreated B
cells at any ratio. These data stronglysuggested that CT and LT
improved the efficiency of B cellsacting as APC and that the
mechanism of this effect was re-lated to the increase in the
intracellular cAMP level, as indi-cated by the effect of
FSK-treated B cells on allogeneic T-cellproliferation.
To analyze the antigen-specific T-cell activation induced
byB-cell antigen presentation, toxin-treated B cells were used
as
APC in the autologous system in order to expand the
antigen-specific T cells of either PPD or TT responder donors.
Theisolated B cells were stimulated with CpG, IL-2, and PPDprotein
or TT, and CT, LT, CT-B, LTK63, or FSK was addedon the same day.
After 3 days, cells were cocultured withautologous PBMC previously
labeled with CFSE. The prolif-eration of PBMC, CD4�, and CD8� T
cells was evaluatedafter 5 days of coculture by FACS analysis. As
shown in Fig. 6,only treatment with CT, LT, or FSK increased
proliferation ofautologous PBMC, CD4�, and CD8� T cells at any
ratio of Bcells to PBMC used, whereas CT-B or LTK63 did not
enhancethe antigen-presenting capacity of B lymphocytes.
In order to check the role of CD86 and HLA-DR in theimprovement
of the APC function of toxin-treated B cells,MLR experiments were
performed in the presence of blockingMAb anti-CD86 and/or MAb
anti-HLA-DR. As expected, thepresence of both blocking reagents
inhibited up to 80% ofPBMC proliferation (Table 1) at ratio of B
cells to PBMC of1:4, indicating the evident contribution of these
molecules inthis system.
CT and LT inhibit TNF-� production while increasing IL-12,
IL-1�, and IL-6 production. To examine the effect of ad-juvants on
the production of cytokines, supernatants from Bcells cultured for
3 days with polyclonal stimuli and treatedwith toxins, FSK, or
nontoxic counterparts or left untreatedwere analyzed by ELISA.
Figure 7 shows the results obtainedfor five donors. Treatment with
CT, LT, or FSK induced asignificant decrease in TNF-� production
(56.3, 120.4, and 64.9pg/106 cells, respectively) compared to the
untreated control(270 pg/106 cells). In contrast, treatment with
CT-B or LTK63
FIG. 5. Proliferation of allogeneic PBMC cocultured with B cells
as APC. Activation of B cells by an increased level of
intracellular cAMPenhances their ability to present alloantigen in
the allogeneic T-cell response. B cells were stimulated with
polyclonal stimuli, including 2.5 �g/mlof CpG ODN 2006, 50 U/ml of
IL-2, and 2 �g/ml of anti-Ig MAb, and simultaneously treated with
the compounds indicated or left untreated (NT)for 3 days and
irradiated. B cells were cocultured with allogeneic PBMC labeled
with CFSE at different B cell/PBMC ratios. After 3 days
ofcoculture, cells were collected and stained with anti-human CD4
or anti-human CD8 MAb. The level of PBMC, CD4�, and CD8�
T-cellproliferation was evaluated by FACS analysis. The error bars
indicate standard deviations for duplicates. Data from three
independent experimentsare shown. PBMC TOT, total PBMC.
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did not significantly influence the cytokine levels (228 and
237pg/106 cells). As shown in Fig. 7, toxin- and FSK-treated B
cellsshowed significant production of IL-12 and IL-1� cytokines.
Inparticular, high levels of IL-1� were detected in toxin-
andFSK-treated samples (1,520, 1,211, and 1,781 pg/106 B
cellstreated with CT, LT, and FSK, respectively), whereas low
butdetectable concentrations of IL-12 were present in B-cell
su-pernatants from CT-, LT-, and FSK-treated samples (260, 243,and
250 pg/106 B cells). Finally, treatment with toxins and FSKinduced
production of amounts of IL-6 larger than that inuntreated B cells
(1,947, 1,542, and 1,273 pg/106 cells for Bcells treated with CT,
LT, and FSK, respectively, versus 970pg/106 cells for untreated B
cells). However, the increase inIL-6 production was not
statistically significant, probably dueto the high variability
among donors. Treatment with CT-B orLTK63 did not result in any
significant difference in IL-12,IL-1�, or IL-6 production compared
to untreated samples.
Effect of adjuvants on sorted CD27� and CD27�
B-cellsubpopulations. Finally, in order to determine if the
differentsubsets of B cells were targeted differently by
adjuvants,CD27� and CD27� B cells were isolated and treated as
de-scribed above for unsorted B cells. In particular, CD86
expres-sion and susceptibility to death were analyzed for the
twodifferent B-cell populations after 3 days of culture. As shown
inFig. 8A, for both subsets a dramatic increase in the level ofCD86
was evident in toxin- or FSK-treated cells compared tothe
corresponding untreated sample, while the presence ofCT-B or LTK63
did not affect CD86 expression. Likewise, forinduction of cell
death, the results indicated that treatmentwith toxins had similar
effects on the two subsets (Fig. 8B).Indeed, at 3 days there was
not a statistically significant differ-ence in the percentages of
Annexin V- and PI-labeled cellsbetween the two subpopulations.
DISCUSSION
The specificity, magnitude, and quality of T-cell-mediatedimmune
responses become conditioned during the early phaseof antigen
presentation. For this reason, analysis of the effectsof adjuvants
on APC could help identify the mechanism ofaction. In the
population of APC that includes DC, B cells, andmacrophages, DC are
the most potent, and for this reason themajority of adjuvant
studies are focused on these cells. We andother authors have
previously shown that CT and LT were ableto mature human DC and
inhibit IL-12 and TNF-� production,whereas CT-B and LTK63 (5, 14,
15) did not have these abil-ities. Several papers described the APC
function of B cells,indicating that in vivo B cells provide extra
and essential anti-gen presentation capacity above that provided by
DC, optimiz-ing expansion and allowing generation of memory and
effectorT cells (9, 10, 17, 18, 23). Therefore, we decided to
investigate
FIG. 6. Antigen-specific T-cell proliferation. Activation of B
cells by an increased level of intracellular cAMP enhances their
ability to presentPPD or TT in the autologous T-cell response. B
cells derived from a PPD or TT responder donor were stimulated with
2.5 �g/ml of CpG ODN2006 and 50 U/ml IL-2 in the presence of PPD or
TT and treated as indicated or left untreated (NT) for 3 days.
Cells were then irradiated andcocultured with autologous PBMC
labeled with CFSE at different B cell/PBMC ratios. After 5 days of
coculture cells were collected and stainedwith anti-human CD4 or
anti-human CD8 MAb. The levels of PBMC, CD4�, and CD8� T cell
proliferation were evaluated by FACS analysis. Theerror bars
indicate standard deviations for duplicates. PBMC TOT, total
PBMC.
TABLE 1. MLR analysis in the presence of blocking MAbsa
B-celltreatment
% of proliferating cells (mean � SD)b
No blocking
Blocking MAbs
Anti-CD86 Anti-HLA-DR
Anti-CD86� anti-
HLA-DR
None 10.17 � 0.81 5.36 � 0.37 3.54 � 0.08 2.07 � 0.31CT 18.00 �
1.02 9.67 � 0.35 4.58 � 0.29 3.61 � 0.42CT-B 11.89 � 1.17 5.90 �
0.21 4.35 � 0.61 2.76 � 0.40LT 20.30 � 0.28 11.42 � 0.43 5.99 �
0.45 3.03 � 0.74LTK63 9.34 � 0.62 5.52 � 0.31 4.43 � 0.04 3.20 �
0.35FSK 18.86 � 0.73 12.00 � 1.12 6.48 � 0.50 6.05 � 0.78
a B cells preincubated with anti-CD86, with anti-HLA-DR, or with
both anti-bodies were not able to induce proliferation of
CFSE-labeled PBMC.
b The percentage of proliferating cells was determined using a B
cell/PBMCratio of 1:4.
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the effects of toxins and their nontoxic counterparts on
theantigen-presenting capacity of human B cells. In the
presentstudy we performed an in vitro comparative evaluation of
theAPC function of human B cells after treatment with CT, CT-B,LT,
LTK63, or FSK, a direct activator of adenylate cyclase, asa
positive control for an increase in intracellular cAMP, ordirectly
with cAMP analogues, such as Db-cAMP and 8Br-cAMP. Our results show
that the enzymatic activity of toxins iscrucial for in vitro
activation of human B cells and improve-ment of their APC capacity.
Indeed, CT and LT, which in-crease intracellular cAMP levels,
induced an evident activa-tion state of human B cells, as judged by
changes in surfacephenotype, whereas none of the enzymatically
inactive de-rivatives of CT or LT tested in this study were able to
modifyactivation markers. In addition, the functional changes in
Bcells, including inhibition of proliferation, susceptibility
tocell death, cytokine production, and an increase in the
an-tigen-presenting capability induced by CT or LT, can bemimicked
consistently by using the pharmacological agonistFSK or cAMP
analogues.
To avoid the high rate of mortality of unstimulated B cellsand
to prolong the in vitro cultures, we decided to performexperiments
in the presence of polyclonal stimuli. These stim-uli, including
antibody to human Ig as an antigen surrogate,CpG as a Toll-like
receptor agonist, and IL-2 as a growthfactor, are required for
activation of both naïve and memory Bcells in the absence of CD4� T
cells (7). Treatment with toxinswas able to increase the expression
of CD86 and HLA class II,confirming results obtained by other
workers (2). Conversely,CT-B and LTK63, even if they were used at
concentrationshigher than those used for the toxins, were unable to
upregu-late the activation markers on human B cells. Recently,
Schnit-
FIG. 8. Effects of CT and LT on CD27� and CD27� B-cell
popu-lations. Toxins act similarly in both subsets in terms of
activation andinduction of cell death. CD27� and CD27� B cells were
sorted byFACSAria, stimulated with polyclonal stimuli, including
2.5 �g/ml ofCpG ODN 2006, 50 U/ml of IL-2, and 2 �g/ml of anti-Ig
MAb, andsimultaneously treated with the compounds indicated or left
untreated(NT) for 3 days. (A) Expression of CD86 was evaluated by
FACSanalysis of CD27� and CD27� B-cell subsets. The histograms show
thepercentages of CD86� cells for CD27� (open bars) or CD27�
(filledbars) B lymphocytes from two donors. The error bars indicate
standarddeviations. (B) Stimulated B-cell subsets, treated as
indicated for 3days, were stained with Annexin V-FITC plus PI and
analyzed byFACS. The percentages of Annexin V-positive and
PI-positive cells areindicated. The error bars indicate standard
deviations.
FIG. 7. Cytokine production. CT and LT strongly inhibit the
production of TNF-� but increase the production of IL-12, IL-1�,
and IL-6. B cellswere stimulated with polyclonal stimuli, including
2.5 �g/ml of CpG ODN 2006, 50 U/ml of IL-2, and 2 �g/ml of anti-Ig
MAb, and simultaneouslytreated with the compounds indicated or left
untreated (NT) for 3 days. B-cell culture supernatants from five
different donors were collected andanalyzed for the presence of the
cytokines indicated by ELISA. The error bars indicate standard
deviations. The asterisks indicate a statisticallysignificant
difference (P 0.05) between the treatment and control (NT)
samples.
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zler et al. showed that treatment of murine B cells with
CT-Binduced a sequence of signaling events related to
cellularactivation and surface molecule expression (37). In
ourstudy, treatment of both unstimulated and polyclonal acti-vated
human B cells with CT-B did not induce any variationsin surface
activation markers and antigen presentation, sug-gesting that there
is a difference in behavior between humanand murine B cells. The
precise mechanism of action ofthese adjuvants has not been
completely elucidated, andthere are controversies concerning the
requirements for androles of the A and B subunits of these toxins
both in vitroand in vivo (reviewed in reference 16). Factors
involved inthe dissimilar findings include the route of
administration,the characteristics of the vaccine antigen,
contaminationof the adjuvant with endotoxin or with holotoxin, and
thespecies of animal used. The difference between the humanand
murine cell responses to nontoxic derivatives of toxinscould be
another important factor that should be taken intoaccount in the
design and development of mucosal adjuvantssuitable for human
vaccination.
It is known that the second messenger cAMP can
haveimmunosuppressive effects on T and B lymphocytes (28, 32,38).
We confirmed these findings, showing that CT, LT, FSK,and cAMP
analogues inhibited the proliferation of B cellsinduced by
polyclonal stimuli. FSK-treated B cells producedlarger amounts of
intracellular cAMP than toxin-treated cells,suggesting that the
difference could represent a possible reasonfor the more pronounced
inhibition of proliferation seen forFSK-treated cells. These
results are in agreement with thoseobtained previously by our group
(39) and by Johnson et al.(21), showing that increased levels of
cAMP were able toinhibit in a dose-dependent manner anti-CD3- or
IL-2-inducedT-cell proliferation. In addition, toxins and FSK made
B cellsmore susceptible to death. Conversely, treatment with CT-B
orLTK63, which lack enzymatic activity, did not alter either
theability of stimulated B cells to proliferate or the induction
ofcell death. These results were expected, since it has beenshown
that cAMP is involved in the regulation of apoptosis inB progenitor
and mature B cells by inducing activation ofprotein kinase A (25,
29), suggesting that physiological ligandsthat control cellular
cAMP levels could play an important rolein the regulation of B-cell
maturation in vivo. Indeed, com-pounds acting on the increase in
intracellular cAMP contentcould have pleiotropic effects on the
immune cells, inducingboth suppressive (inhibition of
proliferation) and stimulatorysignals (activation) at the same
time. The final effect observedin vitro and even more in vivo upon
treatment with toxins isprobably due to a balance of these
signals.
In order to determine if the effects of toxins were
directedmostly toward a particular subset of B cells, such as CD27�
orCD27� populations, we first evaluated the expression of GM1on
gated CD27� and CD27� B cells by using FITC-labeledCT-B. The
results indicated that there was a slightly higherlevel of binding
to CD27� B cells (data not shown). However,when CD86 expression and
induction of cell death after treat-ment with toxins were evaluated
for the two different B-cellpopulations, the results indicated that
CT and LT acted simi-larly in both subsets. Taken together, our
data indicated thatthe toxins induced a real increase in CD86
expression in total
B cells and did not cause selective depletion of the
populationwith a low level of CD86 expression.
Finally, to investigate the effects of the adjuvants on
theantigen-presenting function of human B cells, both allo-MLRand
antigen-specific T-cell proliferation tests were performed.The
ability of B cells treated with CT, LT, or FSK to induceT-cell
proliferation was evident in both the assays. The datacould be
explained by the fact that toxins and FSK were able toinduce an
activation state with upregulation of costimulatorymolecules and
HLA class II, which was responsible for theincreased
antigen-presenting function observed. Indeed, as ex-pected, the use
of blocking MAbs against CD86 and/orHLA-DR in the MLR assay
resulted in a high level of inhibi-tion of PBMC proliferation.
Again, treatment with CT-B andLTK63 did not improve the APC
capacity of stimulated B cells,further confirming the role of cAMP
in the adjuvant activity oftoxins. We and other authors reported
that CT and LT inhibitIL-12 and TNF-� production by human DC,
partially explain-ing the polarization of CD4� T cells toward a Th2
phenotypeobserved when CT- or LT-treated DC were used as APC (5,14,
15). In this study, we detected strong inhibition of
TNF-�production upon treatment of B cells with CT, LT, or
FSK,further supporting the role of cAMP in the modulation ofB-cell
functions. Surprisingly, in contrast to the results for DCtreated
with CT or LT, we observed IL-12 production in toxin-treated B
cells and strong production of IL-1�. As reported inother papers
(reviewed in reference 16), we confirmed that CTand LT induced an
increase in IL-6 production. This peculiarpattern of cytokines
induced by treatment with toxins could beimportant for the
generation of an environment able to drivethe Th1/Th2 polarization
of T cells. Additional studies arerequired to investigate this
issue.
In this work, several observations support the hypothesisthat CT
and LT directly activate human B cells predominantlyby elevating
the intracellular cAMP level. We cannot excludethe possibility that
there is concomitant involvement of otherfactors, including the
signaling induced by binding with theirreceptors. Although in our
model system the presence of en-zymatic activity is required for
the adjuvanticity of toxins, sug-gesting that LTK63 and CT-B do not
act directly on theseAPC, we cannot rule out the possibility that
there is an indirecteffect on APC that is induced by these
compounds in vivo. Ourresults are in agreement with the results of
other studies basedon a requirement for the A subunit of CT for the
induction ofadjuvanticity of B cells. Indeed, it has been
demonstrated thatCTA1-DD, an adjuvant based on the CT A subunit
geneticallylinked to two Ig-binding domains (DD) of staphylococcal
pro-tein A, but not enzymatically inactive mutants, was able
totarget and activate B cells and act as a good mucosal adjuvantin
vivo (1, 12). In this study, our objective was to determine ifthe
enzymatic activity is mandatory for the APC function of Bcells, and
therefore we focused on reagents with 0% or 100%activity. It would
be interesting to check the minimum level ofenzymatic activity
required for adjuvanticity with human Bcells by using recombinant
enterotoxins with greatly reducedenzymatic activity.
Together with our previous data (14, 15), results obtained
inthis study allow us to conclude that the adjuvanticity of
toxins,measured as in vitro activation and the
antigen-presentingability of human APC (both DC and B cells), is
stringently
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correlated to the presence of the enzymatic activity involved
inthe increase in the intracellular cAMP content.
ACKNOWLEDGMENTS
We thank Andrea Cara for suggestions and critical reading of
themanuscript, Laura Pancotto for purification of LT, and
EmanueleFanales-Belasio and Maria Rosaria Pavone-Cossut for
performing theendotoxin analysis.
This study was carried out with financial support from
Commissionof the European Communities Sixth Framework Programme
contractLSHP-CT-2003-503240 (Mucosal Vaccines for Poverty-Related
Dis-eases) (M.T.D.M.) and from grants from the Italian AIDS
NationalProgram (contract 45G/C) (M.T.D.M.).
P.R. and G.D.G. are Novartis employees. The other authors
declareno conflict of interest.
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