Reciprocal Polarization of T and B Cells at
the Immunological Synapse
This information is current as
of June 16, 2017.
Sophie Duchez, Magda Rodrigues, Florie Bertrand and
Salvatore Valitutti
J Immunol published online 19 September 2011
http://www.jimmunol.org/content/early/2011/09/16/jimmun
ol.1100600
http://www.jimmunol.org/content/suppl/2011/09/22/jimmunol.110060
0.DC1
Subscription
Information about subscribing to The Journal of Immunology is online at:
http://jimmunol.org/subscription
Permissions
Email Alerts
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 © 2011 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 16, 2017
Supplementary
Material
Published September 19, 2011, doi:10.4049/jimmunol.1100600
The Journal of Immunology
Reciprocal Polarization of T and B Cells at the Immunological
Synapse
Sophie Duchez, Magda Rodrigues, Florie Bertrand, and Salvatore Valitutti
I
t is well established that the onset of adaptive immune
responses requires the activation of T and B lymphocytes
through the formation of specialized junctions where immune
receptors are engaged with specific Ags. Both T and B cells form
TCR- or BCR-mediated immunological synapses (ISs) that display
common structural and functional properties when interacting with
cells displaying antigenic ligands on their surfaces (1–8).
In both cases, a highly dynamic structure is formed and is
composed of a central supramolecular activation cluster where
TCR or BCR are enriched (site of immunoreceptor internalization
and active signaling) surrounded by a peripheral supramolecular
activation cluster that contains adhesion molecules. In T cells, one
hallmark of IS formation is the Ag-dependent reorientation of
the microtubule-organizing center (MTOC) toward the interacting APC (9–11). This is paralleled by the reorientation of Golgi
apparatus, recycling endosomal compartment (12–15), transmembrane proteins (16), and lytic granules (8, 17) toward the interacting APC or target cell.
A peculiar characteristic of the B cell synapse is that, once
Ags are recognized by BCR, they are internalized, degraded, and
Centre de Physiopathologie de Toulouse Purpan, Section Dynamique Moléculaire
des Interactions Lymphocytaires, INSERM U1043, F 31300 Toulouse, France; and
Université Toulouse III Paul-Sabatier, F 31300 Toulouse, France
Received for publication February 28, 2011. Accepted for publication August 22,
2011.
This work was supported by grants from the Association pour la Recherche sur le
Cancer (Grant SL220100601347) and l’Institut National du Cancer. S.D. was supported by a fellowship from the Fondation pour la Recherche Médicale.
Address correspondence and reprint requests to Salvatore Valitutti, Centre Hospitalier
Universitaire Purpan, INSERM U1043, 31024 Toulouse, Cedex 3, France. E-mail
address: [email protected]
The online version of this article contains supplemental material.
Abbreviations used in this article: BODIPY, 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; CMFDA, 5-chloromethylfluorescein diacetate; CMTMR, 5-(and-6)-(4-chloromethyl)benzoyl)amino)tetramethylrhodamine; DC, dendritic cell; GM130, Golgi
matrix protein of 130 kDa; IS, immunological synapse; MFI, mean fluorescence
intensity; MTOC, microtubule-organizing center; OVAp, OVA peptide; pTyr, phosphotyrosine; SAg, superantigen; TSST-1, toxic shock syndrome toxin-1.
Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1100600
presented (in the form of peptides bound to MHC class II molecules) to Th cells for T cell/B cell cognate interaction (18). Thus,
B cells are capable of forming two kinds of ISs, both central to the
development of adaptive immune responses: one IS for Ag capture
and the other for Ag presentation.
In addition to BCR- and T cell-derived signals, TLR engagement
provides a third central stimulus for B cell activation. Through
TLR signaling, B cells differentiate into specialized cells capable
of communicating with Th cells to undergo Ab diversification,
clonal expansion, and Ig secretion. Various TLR ligands and their
corresponding receptors are responsible for B cell activation and
maturation (19, 20).
The TLR repertoire of human B cells is not fully defined, but it
has been reported that naive human B cells express TLR9 and also
have been shown to be directly activated by CpG (21, 22). Cross
talk between BCR and the TLR9 pathway is supported by recent
observations. It has been reported that BCR-assisted Ag internalization can be instrumental for providing intracellular ligands
for TLR9 and that Ag stimulation in the presence of TLR9 engagement results in enhanced B cell activation (23, 24).
Our hypothesis is that BCR and TLR signaling might influence
B cell capacity to serve as APCs for T cells and that, in turn, T cellderived synaptic signals might enhance B cell activation. T cell/
B cell synapses therefore might serve as a platform for the integration of these three signals central to the outcome of the T cell/
B cell cognate interaction.
In the current study, we investigated the morphological aspects
of the T cell/B cell interaction with a focus on the B cell side of the
IS. In particular, we investigated whether signaling via TLR9 could
synergize with signals derived from BCR in enhancing T cell/B cell
communication at the IS.
We show that Th cells, simultaneously interacting with different
cognate B cells, either naive or previously activated via BCR or
TLR9 engagement, selectively polarized their secretory machinery
toward activated B cells. We also provide the unexpected observation that both naive and preactivated B cells rapidly reoriented
their endocytic/exocytic compartments toward T cells during cognate interactions.
Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017
Cognate interactions between T and B lymphocytes lead to the formation of the immunological synapse (IS) where bidirectional
activation signals are exchanged. Although the molecular architecture and the function of the IS have been studied extensively on the
T cell side, little is known about events occurring during synapse formation in Ag-presenting B cells. We investigated the impact of
BCR and TLR signaling on human B cell activation and on the T and B cell side of the IS. On the T cell side, we observed that T cells
polarized toward both naive and previously activated B cells. Nevertheless, when T cells interacted with different B cells simultaneously, T cells selectively polarized their secretory machinery toward preactivated B cells. Furthermore, both naive and preactivated B cells reoriented their microtubule-organizing center toward the synaptic T cell during cognate interactions. This
phenomenon was rapid and not dependent on T cell secretory activity. Interestingly, not only the microtubule-organizing center
but also the Golgi apparatus and Lamp-3+ and MHC class II+ vesicles all repositioned beneath the IS, suggesting that the entire
endocytic/exocytic B cell compartment was reoriented toward the T cell. Taken together, our results show that the B cell activation
status fine-tunes T cell polarization responses and reveal the capacity of naive and activated B cells to polarize toward T cells
during cognate interactions. The Journal of Immunology, 2011, 187: 000–000.
2
POLARIZATION RESPONSES IN T CELL/B CELL COGNATE INTERACTION
Our results shed new light on T cell/B cell cognate interaction by
showing that the B cell activation status fine-tunes T cell polarization responses and that T and B cells reciprocally polarize for
intercellular cooperation at the IS.
Materials and Methods
Cell isolation
Mice
Six- to 12-week-old C57BL/6 or C57BL/6 OT-II mice and a synthetic
peptide corresponding to amino acid residues 323–339 (ISQAVHAAHAEINEAGR) of OVA (Ovap) were kindly provided by Dr. S. Guerder
(INSERM U1043). All of the mice used were raised and housed under
specific pathogen-free conditions and handled according to protocols approved by the INSERM Ethics Committee on Animal Experimentation in
compliance with European Union guidelines. OT-II T cell lines were
kindly provided by Dr. Eric Espinosa (INSERM U1043)
Measurement of costimulatory molecule expression on T cell or
B cell surfaces by FACS analysis
B cells were either unpulsed or pulsed with 10 ng/ml of the bacterial
superantigen (SAg) toxic shock syndrome toxin-1 (TSST-1) (Toxin
Technology, Sarasota, FL) for 1 h 30 min at 37˚C in culture medium. During
the last 15 min of the pulse, APCs were stained with CellTracker Green 5chloromethylfluorescein diacetate (CMFDA) (Molecular Probes, Eugene,
OR) to allow their discrimination by FACS analysis. CD4+Vb2+ T cells
were loaded with blue 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) 630 (Molecular Probes, Eugene, OR). After being washed, cells were
conjugated by 1 min of centrifugation with CD4+Vb2+ T cells at a ratio of
one T cell per B cell in 200 ml culture medium in 96-well U-bottom
microplates. After overnight culture, T cell/B cell conjugates were broken in EDTA/PBS, and cells were stained with either anti-CD40 mAb
(clone 5C3; BD Biosciences), anti-CD54 mAb (clone HA58; BD Biosciences), anti-CD80 mAb (clone L307.4; BD Biosciences), anti-CD86
(clone B70/B7-2; BD Biosciences), or anti-HLA-DR mAb (clone L243
supernatant HB-55; American Type Culture Collection, Manassas, VA).
Primary Abs were followed by PE-labeled goat anti-mouse isotype-specific
Ab (Molecular Probes).
In some experiments, naive and preactivated B cells were pulsed with
10 ng/ml TSST-1 for 1 h 30 min at 37˚C in culture medium. During the last
TCR downregulation
B cells were either unpulsed or pulsed with different concentrations of
TSST-1 for 1 h 30 min at 37˚C in culture medium. During the last 15 min of
pulsing, APCs were stained with CellTracker Orange CMTMR (Molecular
Probes) to allow their discrimination by FACS analysis. CD4+Vb2+ T cells
were loaded with BODIPY 630. After being washed, cells were conjugated
by 1 min of centrifugation with CD4+Vb2+ T cells at a ratio of one T cell
per B cell in 200 ml culture medium in 96-well U-bottom microplates.
After 3 h of conjugation, T cell/B cell conjugates were broken in EDTA/
PBS, and cells were stained with FITC-labeled anti-Vb2 mAb. Analyses of
cell surface expression of TCRVb2 on CD4+ T cells were performed on
a FACSCalibur apparatus.
Measurement of IFN-g production by FACS analysis
B cells were either unpulsed or pulsed with 10 ng/ml TSST-1 for 1 h 30 min
at 37˚C in culture medium. During the last 15 min of pulsing, APCs were
stained with CMFDA to allow their discrimination by FACS analysis.
T cells were loaded with BODIPY 630. Cells were conjugated with CD4+
Vb2+ T cells at a 1:1 ratio in 96-well U-bottom microplates. Brefeldin A
(Sigma-Aldrich, St. Louis, MO) was added to the culture at a final concentration of 10 mg/ml to stop secretion. After 3 h of conjugation, T cell/
B cell conjugates were broken in EDTA/PBS, and cells were fixed, permeabilized with 0.1% saponin (in PBS containing 3% BSA/HEPES), and
stained with PE-labeled anti-IFN-g mAb (clone B27; BD Pharmingen, San
Diego, CA). Analyses were performed on a FACSCalibur apparatus.
Intracellular staining for confocal microscopy
B cells were either unpulsed or pulsed with 10 ng/ml TSST-1 and were
stained with CMFDA, CMTMR, or BODIPY 630. At different times
after conjugation, cells were resuspended gently and seeded on poly-Llysine–coated coverslips. Cells were fixed in PBS containing 3% paraformaldehyde, permeabilized with 0.1% saponin (in PBS containing
3% BSA/HEPES), and labeled for 45 min with the following primary
Abs: anti-phosphotyrosine (pTyr) mAb (clone sc-7020; Santa Cruz Biotechnology, Santa Cruz, CA), anti a-tubulin mAb (clone DM1A; SigmaAldrich), anti-Golgi matrix protein of 130 kDa (GM130) mAb (clone 35/
GM130; BD Biosciences), anti-HLA-DR mAb (clone L243 supernatant
HB-55, American Type Culture Collection), anti-CD63 mAb (clone MEM259; Abcam), followed by secondary goat anti-mouse isotype-specific Ab
or goat anti-rabbit labeled with Alexa Fluor 350, Alexa Fluor 488, Alexa
Fluor 546, or Alexa Fluor 647 (Molecular Probes).
Before fluorescent transferrin incorporation, B cells were incubated for
1 h 30 min at 37˚C in serum-free medium to deplete the intracellular
compartments of transferrin and then incubated for 60 min with 50 mg/ml
transferrin A546 (Invitrogen) at 37˚C in culture medium.
Slides were washed and mounted in 90% glycerol/PBS containing 2.5%
1,4-diazabicyclo[2.2.2]octane (Fluka) and examined using a LSM 710
inverted confocal microscope (Zeiss, Oberkochen, Germany) equipped with
a Plan Apochromat 363 1.4 numerical aperture oil immersion objective
and with an electronic zoom of 3. For each pair of Abs used, standardized
conditions for pinhole size and for gain and offset (brightness and contrast)
were used for image capture. For the given settings, staining with secondary Ab only did not result in detectable fluorescence.
Image quantification
Scoring of the slides was performed in a blinded fashion by evaluating for
each condition at least 50 T cell/B cell conjugates in randomly selected
fields from at least three independent experiments.
To evaluate the polarization of the secretory machinery toward the IS,
distances of MTOC from the center of the T cell/B cell contact site were
Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017
Human T and B cells were isolated from whole blood of healthy donors
(Centre Hospitalier Universitaire Purpan, Toulouse, France). Blood samples
were obtained following standard ethical procedures (Helsinki protocol) and
with the approval of the concerned Internal Review Boards.
The CD4+ T cell fraction was sorted using RosetteSep (StemCell
Technologies, Vancouver, BC, Canada). CD4+Vb2+ T cells were isolated
by positive selection using an anti-Vb2 Ab (clone MPB2D5; Beckman
Coulter, Fullerton, CA) and goat anti-mouse IgG microbeads (Miltenyi
Biotec, Auburn, CA). Cell purity was checked by FACS analysis on a
FACSCalibur (BD Biosciences, San Jose, CA), and CD4+Vb2+ fractions
were routinely 92–98% pure. CD4+Vb2+ T cells were expanded in vitro in
RPMI 1640 medium, 5% human serum, and IL-2 (250 U/ml) in the
presence of anti-CD3/CD28 mAb-coated Dynabeads (Invitrogen, Carlsbad, CA) at a ratio of one bead per T cell. T cell lines were used between 2
and 4 wk of culture.
A pre-enrichment of human CD19+ B cells was obtained using RosetteSep (StemCell Technologies). Naive B cells were isolated by negative
selection using the naive B cell isolation kit II (Miltenyi Biotec). Cell
purity was assessed by FACS analysis using PE-labeled anti-CD27 mAb
(clone O323; eBioscience, San Diego, CA) and FITC-labeled anti-CD19
mAb (clone HIB19; eBioscience). The percentage of naive CD19+CD272
B cells was routinely 95–99% pure. Freshly isolated naive B cells were
cultured in IMDM and 10% FCS (culture medium). Part of the sample was
preactivated in vitro at a concentration of 1 3 106 B cells per milliliter in
24-well plates for 48 h with 2.5 mg/ml F(ab9)2 goat anti-human IgM
(Jackson ImmunoResearch, West Grove, PA), 2.5 mg/ml of TLR9-selective
agonist CpG ODN 2006 (59-TCgTCgTTTTgTCgTTTTgTCgTT-39; CaylaInvivoGen), or both reagents. Mouse CD4+ T cells were isolated from
C57BL/6 OT-II mice using the mouse CD4+ T Cell Isolation Kit (Miltenyi
Biotec). Mouse resting B cells were isolated from C57BL/6 mice by
negative selection using CD43 microbeads (Miltenyi Biotec). Cell purities
of the different purifications were checked by FACS analysis on a FACSCalibur and were routinely 92–99% pure. Cells were cultured in RPMI
1640 medium containing 5% FCS.
15 min of the pulse, naive and preactivated B cells were stained with
CMFDA or 5-(and-6)-(4-chloromethyl)benzoyl)amino)tetramethylrhodamine
(CMTMR), respectively, to allow their discrimination by FACS analysis.
After being washed, naive B cells were cocultured with CD4+Vb2+ T cells
and with preactivated B cells at a 1:1:1 ratio in 96-well U-bottom
microplates. After 16 h of coculture, T cell/B cell conjugates were broken, and cells were stained with a biotinylated anti-CD4 mAb (clone RPAT4; BD Biosciences) and either anti-CD54 mAb, anti-CD80 mAb, antiCD86 mAb, or anti-CD69 mAb. Primary Abs were followed by Alexa
Fluor 633-labeled streptavidin (Molecular Probes) and PE-Cy5.5-labeled
goat anti-mouse isotype-specific Ab (Molecular Probes). In additional
experiments, naive B cells were cultured either in the presence or in the
absence of CD4+Vb2+ T cells for 4 h before the addition of preactivated
B cells at 1:1:1 ratio. Staining was performed as described above. Analyses
were performed on a FACSCalibur.
The Journal of Immunology
measured on unprocessed images, using the Profile function of the Zeiss
software (Supplemental Fig. 1A).
To evaluate the position of GM130, transferrin+, and CD63+ vesicles in
relation to a-tubulin staining, unprocessed images were analyzed using
the Region Measurements function of the Metamorph software (Universal
Imaging, Downingtown, PA). Two fixed regions were drawn on each
B cell, one containing MTOC and the other containing the entire cell
(Supplemental Fig. 1B). The software calculates the integrated fluorescence intensity in both regions. For each conjugate, a ratio between the
fluorescence in the MTOC region and the fluorescence in the entire B cell
region was calculated. Results expressed the percentage of staining detected in the MTOC area per cell.
To evaluate the distance of the major intracellular pool of MHC class II
from the IS, distances of the vesicles from the center of the T cell/B cell
contact site were measured on unprocessed images, using the Profile
function of the Zeiss software (Supplemental Fig. 1C).
Time-lapse confocal microscopy
Results
Minor impact of B cell activation status on T cell responses
We investigated the possible impact of different signals (BCR
cross-linking, TLR9 signaling, or combined signals) on human
peripheral B cell activation and their impact on the formation of
a productive IS with human CD4+ Th cells.
In the first set of experiments, we investigated by FACS analysis the surface expression of accessory molecules and activation
markers on B cells, either naive or previously activated using either
an anti-m Ab (for BCR cross-linking), the TLR9 agonist CpG
ODN 2006, or both reagents. For anti-m stimulation, increased
surface expression of CD86, CD40, and MHC class II molecules
was observed. For CpG treatment, increased surface expression of
CD54, CD69, CD80, CD86, CD40, and MHC class II was observed. The treatment with the two stimuli had a synergistic effect
on the upregulation of the above markers (except CD40) (Fig. 1A).
Overnight coculture with CD4+Vb2+ Th cells induced a significant increase in accessory molecule and activation marker expression in B cells pulsed with the bacterial SAg TSST-1, indicating that coculture with Th cells has a strong impact on B cell
activation (Fig. 1A). It should be noted that this important upregulation of activation molecules on TSST-1–pulsed B cells was
seen only in the presence of T cells (Fig. 1A), indicating that
TSST-1 does not have by itself an effect on B cell activation.
In the second set of experiments, we investigated the effect of
B cell preactivation on T cell responses. We measured, by flow
cytometry, the downregulation of Vb2 TCR and the production of
IFN-g in T cells after 3 h of culture with naive or preactivated
B cells. As shown in Fig. 1B, T cells underwent SAg dosedependent TCR downregulation and IFN-g production that were
increased only moderately by preactivation of presenting B cells.
Accordingly, B cell activation status did not strongly influence the
upregulation of CD54 and CD69 in CD4+ T cells after overnight
culture with B cells (Fig. 1C).
These results indicate that, although BCR, TLR, and T cellderived signals can either individually or in combination enhance
B cell activation, the B cell activation status does not substantially
influence T cell responses.
Activation status of B cells modulates T cell polarization
responses at the IS
We next investigated the polarization of the T cell tubulin cytoskeleton toward the IS formed with B cells at different states of
activation by confocal microscopy. Th cells were cocultured for 30
min with B cells either unpulsed or pulsed with TSST-1, fixed,
permeabilized, and stained for a-tubulin and phosphotyrosine
(pTyr). T cells, interacting with pulsed B cells, polarized their
secretory machinery toward B cells (independently of whether
they were untreated or previously activated), as detected by pTyr
staining at the T cell/B cell contact site and reorientation of the
MTOC beneath the synaptic area (Fig. 2A).
Measurement of the distances between the center of the IS and
the T cell MTOC in a large number of conjugates showed that T cell
MTOC polarization toward B cells occurred to a similar extent
regardless of B cell activation status (Fig. 2A, bottom panels).
Similar results were observed when T cell polarization was monitored 5 min after T cell/B cell conjugate formation (data not
shown). The approach used for the measurement of the T cell
MTOC distance from the IS is depicted in Supplemental Fig. 1A.
Having observed that Th cells similarly polarized toward naive
or preactivated B cells encountered individually, we asked what
would be the behavior of Th cells during simultaneous interaction
with B cells displaying different activation states.
Th cells were cocultured with TSST-1–pulsed B cells, either
untreated or preactivated (with either anti-BCR or CpG). After
fixation, permeabilization, and staining, polarization responses
were investigated in T cells found to be simultaneously in contact
with one naive and one preactivated B cell. As shown in Fig. 2B,
in randomly selected three-cell conjugates, T cells preferentially
polarized toward B cells that were activated previously.
Taken together, the above results indicate that, on one hand,
T cells polarize toward B cells encountered individually regardless
of their activation status. On the other hand, in T cells simultaneously interacting with B cells displaying different activation
states, polarization responses are directed preferentially toward
activated B cells.
Rapid B cell polarization responses toward T cells at the IS
An intriguing observation made when studying T cell polarization
toward B cells was that the B cell MTOC (in both naive and
preactivated B cells) also polarized toward the opposing T cell
(Fig. 2, Supplemental Video 1).
B cell polarization was rapid, because comparable polarization
responses were detected in B cells (both naive and preactivated) 5
(Fig. 3A) or 30 min (Fig. 3B) after conjugation.
It should be noted that measurements of the MTOC distance
from the IS in unpulsed B cells were shorter in naive B cells when
compared with those in activated B cells. This observation is due to
the fact that activated B cells are larger. It also should be noted that
measurement of the B cell MTOC distances from the IS in activated
B cells (in particular when activated with both signals) showed
reduced polarization responses of these cells when compared with
those of naive B cells. Measurements were performed as shown in
Supplemental Fig. 1A.
Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017
Naive B cells were pulsed with 10 ng/ml TSST-1 and were stained with
CMTMR to allow their discrimination. Alternatively, T cells were stained
with BODIPY 630. After being washed, T and B cells were loaded with
Tubulin Tracker (Oregon Green 488 Taxol, bis-acetate, Molecular Probes).
B cells were seeded into microchambers (Lab-Tek Chamber coverglass,
Nalge Nunc International, Naperville, IL) previously coated with poly-Dlysine (Sigma-Aldrich). Measurements were started after the addition of
T cells. In parallel experiments, naive B cells and preactivated B cells were
pulsed with 10 ng/ml TSST-1 for 1 h 30 min at 37˚C in culture medium.
During the last 15 min of pulsing, CMTMR was added to the culture
medium of naive B cells, and BODIPY 630 was added to the culture
medium of preactivated B cells to allow their discrimination. After being
washed, T and B cells were loaded with Tubulin Tracker. Naive B cells
were seeded into microchambers previously coated with poly-D-lysine.
Measurements were started after the addition of T cells. T cells and naive
B cells were cocultured for 1 h followed by the addition of preactivated
B cells to the microchamber.
Fluorescence measurements were done on a LSM 710 confocal microscope at 37˚C, 5% CO2. Image sequences of the time-lapse recording
were processed using the ZEN software.
3
4
POLARIZATION RESPONSES IN T CELL/B CELL COGNATE INTERACTION
It is interesting to note that B cell polarization was not linked to
T cell activation and polarization responses. Indeed, pretreatment
of Th cells with monensin (to block secretion) or colchicine (to
disrupt microtubule structure) had a limited effect on B cell polarization responses toward the IS (Supplemental Fig. 2).
We next used time-lapse video microscopy to monitor the dynamics of tubulin polarization in live T cell/B cell conjugates as
detected by the MTOC reorientation toward the IS. To this end, Th
cells and TSST-1–pulsed naive B cells loaded with Tubulin Tracker
were visualized (Fig. 4, Supplemental Videos 2, 3). This approach
illustrated that, when naive B cells pulsed with TSST-1 entered
into contact with T cells, they underwent rapid MTOC reorientation toward the interacting T cells. This event was concomitant
with the previously described T cell MTOC repolarization (25). No
Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017
FIGURE 1. Functional outcome of human CD4+Vb2+ T cell/B cell interaction. A, CD54, CD69, CD40 CD80, CD86, and MHC class II expression
measured by FACS analysis on B cells either naive (blue) or preactivated with anti-m Ab (purple), with CpG (yellow), or with anti-m Ab and CpG (light
blue) for 2 d. B cells were either unpulsed or pulsed with 10 ng/ml TSST-1 and were cultured overnight either in the presence or in the absence of Th cells at
a 1:1 ratio as indicated. T cells were excluded from the analysis on the basis of BODIPY 630 fluorescence. Histogram bars represent SEM. Data are
expressed as mean fluorescence intensity (MFI) fold increase. Data are from at least three independent experiments using cells from three different donors.
B, Naive or preactivated B cells (either unpulsed or pulsed with TSST-1) and loaded with either CMTMR (for TCR staining) or CMFDA (for IFN-g
staining) were conjugated with Th cells loaded with BODIPY 630 at a 1:1 ratio. To measure TCR downregulation, cells were stained with FITC-labeled
anti-Vb2 TCR after 3 h of incubation at 37˚C (left panel). B cells were excluded from the analysis on the basis of CMTMR red fluorescence. Data are from
four independent experiments performed using cells from four different donors for B cells and three different donors for Th cells. Statistical significance of
the difference between the different conditions of B cell stimulations was evaluated by a two-way ANOVA test using GraphPad Prism software. ***p ,
0.0001. To measure intracellular IFN-g production, cells were fixed, permeabilized, and stained with PE-labeled anti-IFN-g after 3 h of incubation at 37˚C
(right panel). B cells were excluded from the analysis on the basis of CMFDA green fluorescence. No statistical significance of the difference between the
different conditions of B cell stimulations, evaluated by a two-way ANOVA test using GraphPad Prism software, was found. Data are from four independent
experiments performed using cells from four different donors for B cells and three different donors for Th cells. C, CD54 and CD69 cell surface expression
was measured by FACS analysis on Th cells after an overnight culture. Histogram bars represent SEM. Data are expressed as MFI-fold increase on T cells.
Data are from three to four independent experiments performed using cells from three different donors.
The Journal of Immunology
5
Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017
FIGURE 2. The activation status of B cells modulates T cell polarization responses at the IS. A, B cells either naive or preactivated with either anti-m Ab,
CpG, or both reagents for 2 d were pulsed with 10 ng/ml TSST-1 (or left unpulsed) and loaded with CMTMR (red). B cells were conjugated for 30 min with
Th cells at a 1:1 ratio. Cells were fixed, permeabilized, and stained for a-tubulin (green) and pTyr (blue). Insets show differential interference contrast
images merged with pTyr staining represented in a pseudocolor scale. Lower panels show the distances between the center of the IS and the T cell MTOC
measured using the Profile function of the Zeiss software. Each dot corresponds to a T cell/B cell conjugate; the number of scored conjugates ranged
between 75 and 102 for the different conditions. Data are from three independent experiments performed using cells from three different donors. Statistical
significance of the difference between groups was evaluated by an unpaired Student t test using GraphPad Prism software. The numbers in red indicate
mean distance. ***p , 0.001. B, Naive B cells and B cells preactivated with anti-m Ab or CpG were pulsed with 10 ng/ml TSST-1 and loaded with
CMTMR (naive B cells, red) or with CMFDA (preactivated B cells, green). Cells were conjugated simultaneously for 30 min with Th cells at a 1:1:1 ratio.
Cells were fixed, permeabilized, and stained for a-tubulin (blue). Distances between the centers of the two ISs and the T cell MTOC were measured using
the Profile function of the Zeiss software. Each dot corresponds to a T cell/B cell conjugate. A total of 87 three-cell conjugates (left panels) and 51 threecell conjugates (right panels) were scored from three independent experiments performed using cells from different donors. Statistical significance of the
difference between groups was evaluated by an unpaired Student t test using GraphPad Prism software. ***p , 0.001.
6
POLARIZATION RESPONSES IN T CELL/B CELL COGNATE INTERACTION
preferential order for T cell or B cell MTOC repolarization toward
the contact zone was seen in different videos.
We also investigated whether Th cells, already polarized toward
naive B cells, could repolarize their MTOC toward new contact
sites formed with preactivated B cells. As shown in Supplemental
Videos 4 and 5, in T cell/naive B cell conjugates, MTOC reorientation toward the incoming preactivated B cells readily occurred,
indicating, in line with our previously reported data, that T cell
polarization responses can be readjusted rapidly (25, 26).
Interestingly, this competition among B cells resulting in preferential polarization of Th cell secretory machinery toward the
preactivated B cells did not affect the upregulation of accessory
molecules and activation markers on naive B cell surfaces (neither
if preactivated B cell were added at the time 0 of the culture nor if
they were added 4 h later; Supplemental Fig. 3). These results are in
agreement with previously published data showing that the upregulation of surface activation markers by dendritic cells (DCs)
requires neither dedicated Th cell polarization nor Th cell/DC
contact (27–29).
The above results show that, upon cognate interaction, both Th
cells and B cells reciprocally polarize their tubulin cytoskeletons
toward the IS. They also furnish further experimental support to the
notion that T cell polarization responses are endowed a high degree
of plasticity.
B cell polarization in Ag-specific T cell/B cell conjugates
The above-reported results showed remarkable B cell microtubule
reorientation toward the IS. These observations raised the question
FIGURE 4. Time-lapse video microscopy of MTOC dynamics in live B cells and Th cells. A sequence of snapshots depicting reciprocal reorientation of
the MTOC in a naive B cell interacting with a CD4+ T cell is shown. Untreated naive B cells pulsed with 10 ng/ml TSST-1 and loaded with CMTMR (red)
were conjugated with CD4+Vb2+ T cells. Both cells were loaded with the Tubulin Tracker (green). Bottom panels show tubulin staining in a pseudocolor
scale. Data are from one representative experiment out of four.
Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017
FIGURE 3. Both naive and activated B cells polarize toward T cells at the immunological synapse. The distances between the center of the IS and the B
cell MTOC were measured using the Profile function of the Zeiss software after 5 min of conjugation (A) or 30 min of conjugation (B). Each dot corresponds to a T cell/B cell conjugate. The number of scored conjugates ranged between 60 and 93 for the different conditions at 5 min and between 75 and
102 for the different conditions at 30 min. Data are from three independent experiments performed using cells from different donors. Statistical significance
of the difference between groups was evaluated by an unpaired Student t test using GraphPad Prism software. The numbers indicate mean distance. **p ,
0.01, ***p , 0.001.
The Journal of Immunology
of whether this phenomenon could be linked to the SAg-mediated
stimulation or also could be seen in Ag-specific T cell/B cell
conjugates in a mouse model. To address this question, MTOC and
pTyr staining were investigated at the IS formed between OT-II
CD4+ mouse T cells (which carry a transgenic TCR specific for
an OVA-derived peptide) and normal resting mouse B cells, unpulsed or pulsed with Ovap. As shown in Fig. 5, cognate interactions between mouse resting B cells and Th cells resulted in
pTyr staining at the IS and in reciprocal polarization of the T and
B cell MTOCs toward the IS.
These results indicated that B cell polarization responses toward
the IS are not linked to SAg stimulation and are not peculiar to
human B cells, being seen in mouse Ag-specific T cell/B cell
conjugates.
Endocytic/exocytic compartments of B cells polarize toward
T cells
FIGURE 5. B cell polarization is seen in Agspecific T cell/B cell conjugates. A, Mouse naive
B cells either unpulsed or pulsed with 10 mM Ovap
were loaded with CMTMR (red) and were conjugated for 30 min with CD4+ OT-II T cells at a 1:1
ratio. Cells were fixed, permeabilized, and stained
for a-tubulin (green) and pTyr (blue). Right panels show differential interference contrast images
merged with pTyr staining represented in a pseudocolor scale. B, Distances between the center of the
IS and the B cell MTOC (left panel) or between the
center of the IS and the T cell MTOC (right panel)
were measured using the Profile function of the
Zeiss software. Each dot corresponds to a T cell/
B cell conjugate. A total of 88 conjugates and 76
conjugates were scored from three independent
experiments for unpulsed and pulsed B cells, respectively. Experiments were performed using cells
from three different mice for B cells and from two
different mice for Th cells. Statistical significance of
the difference between groups was evaluated by an
unpaired Student t test using GraphPad Prism software. ***p , 0.001.
expressed high levels of MHC class II molecules on their surfaces. This was even more difficult in naive B cells, because only
a fraction of these cells (∼50%) exhibited visible intracellular
MHC class II+ vesicles (Fig. 6A, upper panels). We measured the
distance of the major B cell MHC class II+ intracellular pool
(when it was detectable) from the IS as shown in Supplemental
Fig. 3C. This analysis showed that in both naive and preactivated
B cells MHC class II+ vesicles translocated toward the synaptic
area (Fig. 6A).
We also investigated the localization of CD63+ and transferrin+
vesicles in pulsed and unpulsed B cells as well as their position
in relation to MTOC localization. This analysis showed that, in
both pulsed and unpulsed B cells, a significant fraction of CD63+
vesicles localized in proximity to the MTOC (Fig. 6B). These
results indicated that a large part of the lysosomal/endosomal
compartment is associated with the microtubules and moves with
the MTOC toward the IS during cognate interactions. Loading
of B cells with transferrin gave similar results. Although in naive
B cells, transferrin+ compartments were not detectable, in activated B cells ∼90% of transferrin+ vesicles were detected in
proximity to the MTOC, in both unpulsed B cells and B cells
having polarized toward the cognate Th cells (Fig. 6B).
Together, the above results indicate that, in B cells, the secretory
compartment, the endosomal/lysosomal compartments, and MHC
class II+ vesicles are located in proximity to the MTOC and are
repositioned toward the opposing Th cell upon IS formation.
Discussion
In the current study, we used human primary B cells and Th cells to
investigate the impact of B cell activation via BCR or TLR9 on
cellular cooperation at the IS.
We report that the activation status of B cells modulates Th cell
polarization responses by biasing T cell polarization toward B cells
that have been activated previously. Our results also reveal that both
Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017
Having observed that the B cell MTOC rapidly polarized toward
the synaptic T cell, we asked whether the endocytic/exocytic
compartments of B cells also repolarized toward the IS. To this
end, four-color staining was used to visualize, by confocal microscopy, the tubulin cytoskeleton, Golgi apparatus, and MHC
class II+ vesicles in human B cells conjugated with cognate
T cells. As shown in Fig. 6A, this approach showed that, in both T
and B cells, the Golgi apparatus (as detected by staining with the
Golgi protein GM130) and MTOC moved together toward the
IS, indicating that both cells reciprocally polarized their secretory machinery toward each other. Scoring of randomly selected
B cells conjugated with Th cells (as depicted in Supplemental Fig.
1B) showed that ∼90% GM130 staining was detected in proximity
to the MTOC in unpulsed or TSST-1–pulsed B cells, indicating
that these compartments move together during the polarization
process (Fig. 6A). Quantification of MHC class II+ vesicle localization in relation with the MTOC was complex, because B cells
7
8
POLARIZATION RESPONSES IN T CELL/B CELL COGNATE INTERACTION
Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017
FIGURE 6. The entire endocytic/endosomal compartment of B cells polarizes toward T cells. A, Naive B cells either unpulsed or pulsed with TSST-1
(upper panels) and B cells previously activated with anti-m Ab and CpG (either unpulsed or pulsed with TSST-1, lower panels) were loaded with CMFDA
(green) and were conjugated for 1 h with CD4+Vb2+ T cells at a 1:1 ratio. Cells were fixed, permeabilized, and stained for a-tubulin (cyan), GM130 (red),
and MHC class II (blue). Quantification of morphological data is shown in right panels. Scoring was performed using the Profile function of the Zeiss
software and the Region Measurements function of the Metamorph software as indicated in the Materials and Methods section. The number of scored
conjugates ranged between 26 and 65 for the different conditions. Statistical significance of the difference between groups was evaluated by an unpaired
Student t test using GraphPad Prism software. ***p , 0.001. B, Cells loaded with transferrin (red) were fixed, permeabilized, and stained for a-tubulin
(cyan) and Lamp-3 (blue). Quantification of morphological data is shown in the right panels. Scoring was performed using the Region Measurements
function of the Metamorph software as indicated in the Materials and Methods section. The number of scored conjugates ranged between 35 and 64 for the
different conditions. Data are from three independent experiments performed using cells from three different donors. In all of the different conditions
analyzed, MTOCs of B cells polarized toward the T cells in agreement with data in Fig. 3 (data not shown).
The Journal of Immunology
It has been shown previously that the APC side of the IS also is an
active site of molecular rearrangement and signaling. It has been
reported that lipid raft and MHC class II molecules displaying
specific antigenic peptides are enriched at the T cell/APC IS (35,
36) and that a functional actin cytoskeleton in DCs is required for
efficient conjugation with naive T cells during Ag presentation
(37). Moreover, in DCs, CD80 clusters have been shown at the IS
in parallel with CD28 clustering on T cell surfaces (38). Finally,
a recent study showed that, in murine TLR-activated DCs interacting with CD8+ T cells, the DC MTOC is found to be enriched
at the contact site with T cells, leading to polarized secretion of
IL-12 (39). Our results are in agreement with and extend these
findings by showing that B cells (independently of their activation
status) polarize toward T cells at the IS and that these polarization
responses are as rapid as those of T cells.
The observation of rapid B cell polarization responses (that in
some cases preceded T cell polarization) suggests that these responses are not dependent on the synaptic delivery of secreted
mediators (such as cytokines and/or chemokines) by T cells. In
support of this hypothesis, we show that B cell polarization toward
the IS also can occur in conditions in which T cell secretion at the
IS is blocked.
What could be the functional role of these rapid B cell polarization responses?
One possibility is that, in particular, in previously activated
B cells, the secretion of B cell cytokines (40, 41) and/or chemokines (42) might modulate IS stability and T cell activation. Thus,
by analogy to recent reports in DCs (39), B cell polarization might
be at work to enhance T cell activation at the IS. An additional
possibility is that the capacity of naive and activated B cells to
rapidly polarize their secretory and Ag-processing machinery toward T cells might optimize the delivery of antigenic ligands to
the cell–cell contact site, therefore making Ag presentation by
B cells more efficient. In other words, B cell polarization might be
instrumental to optimize the “the immunological relay race” that
is central to T cell/B cell cooperation (1, 43, 44). Our results are
certainly descriptive and do not allow us to establish a mechanistic
link between B cell polarization toward the IS and efficiency of
Ag presentation to T cells. However, it is tempting to speculate
that, during T and B cell motion through lymphoid organs, with
the formation of transient and dynamic contacts, the observed
rapid B cell repolarization responses might be instrumental to
facilitating the delivery of antigenic ligands to the synaptic area
for TCR recognition.
An intriguing observation that came from the inspection of
a large number of T cell/B cell conjugates is that B cell polarization responses tend to be less “complete” than those of T cells
(Supplemental Video 1, also compare the measurements of the T
and B cell MTOC distances from the IS in Figs. 2 and 3). Thus,
it appears that, although the T cells completely polarize their
MTOCs toward B cells, B cells move their secretory and Agpresenting machinery toward the T cell without reaching complete polarization. The mechanism and the functional role of
these incomplete B cell polarization responses are elusive. Nevertheless, these observations support the hypothesis that T and
B cell polarization responses might be under the control of different mechanisms.
All in all, our results shed new light on the fine-tuning of the
T cell/B cell cognate interactions and provide evidence for Agpresenting B cell polarization toward T cells at the IS, revealing bidirectional selectivity of polarization during cell–cell cooperation. Further research is required to define the molecular
mechanisms of these polarization responses and their functional
impact on the outcome of T cell/B cell cooperation.
Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017
naive and preactivated B cells rapidly reoriented their MTOC,
Golgi apparatus, MHC class II compartment, and recycling endosomal compartment toward the IS.
Although our study does not investigate in detail the functional
consequences of the repositioning of the B cell endocytic compartment toward the IS, it describes surprising rapid B cell reactivity upon encounter with cognate T cells that might be involved
in Ag presentation and is worthy of in-depth study in the future.
We have shown previously that Th cells form dynamic ISs with
APCs and that, when interacting simultaneously with APCs offering different antigenic stimuli, they selectively polarize their
secretory machinery toward the APC offering the strongest stimulus, via a protein kinase Cz-dependent mechanism (25, 27). This
phenomenon is instrumental for selective activation of cognate
DCs (27, 30) and can play a central role in dedicated help delivery
to B cells (31–33). In this article, we extend our previous results
by showing that activation of B cells, via either innate or adaptive
receptors, can orient T cell “choice”. We also provide further
experimental evidence that T cell polarization is endowed a high
degree of plasticity that allows Th cells to rapidly adjust their
polarization responses toward the APC providing the strongest
stimulus (25, 26). It is tempting to speculate that, in the course of
an immune response, TLR and/or BCR triggering by pathogenderived products might predispose B cells to receive T cell help,
thus contributing to tight control of B cell activation and differentiation in Ab-secreting cells. The molecular mechanisms of the
observed impact of B cell activation on T cell polarization responses are presently elusive. We hypothesize that the activation
of B cells via TLR and/or BCR might induce a global increase
of MHC class II, adhesion, and/or costimulatory molecules on
B cells that could help to orient T cell polarization responses.
It is interesting to consider these results in parallel with our
present observation that, for human B cell activation, signals derived from T cells are more powerful than signals derived from
BCR and TLR9. Indeed, although pretreatment of B cells with antiBCR and/or TLR9 ligand had a detectable but modest effect on
B cell activation, coculture with cognate Th cells resulted in
dramatic expression of B cell surface activation markers (Fig. 1A).
We speculate that, in the course of T cell/B cell cooperation,
a positive activation loop might take place at the interface between
Th cells and Ag-presenting B cells. The preactivation of B cells
via innate or adaptive immune receptors might favor IS formation
and help delivery by T cells; help delivery, in turn, might further
activate Ag-presenting B cells, making them suitable partners for
new productive encounters with Th cells.
Our results also show that stable Th cell polarization toward
naive B cells is not required for the upregulation of activation
markers by B cells. These results are in agreement with previously
reported data showing that T cell signals, for the upregulation of
accessory molecules and activation markers, can be delivered in
a bystander fashion to DCs, whereas dedicated Th polarization is
required for DC activation of IL-12 production (27, 30). It is
tempting to speculate that, by analogy with DCs, different B cell
responses such as proliferation, Ab class switch, and/or secretion
might be more strictly dependent on Th cell-dedicated polarization (31).
Our results also shed new light on the B cell side of the IS. The
mechanisms in T cells translating the IS molecular dynamics into
biological responses are fairly understood (34). Conversely, the
impact of IS formation on Ag-presenting B cell biology remains to
be elucidated. By showing that SAg-pulsed as well as Ag-pulsed
B cells orient their secretory and Ag-processing apparatus toward
the synaptic Th cell, we identify a new mechanism of B cell
participation in intercellular cooperation.
9
10
POLARIZATION RESPONSES IN T CELL/B CELL COGNATE INTERACTION
Acknowledgments
We thank Loic Dupre, Eric Espinosa, and Nicolas Fazilleau for discussion
and critical reading of the manuscript and Cedric Mongellaz for discussion
and help with flow cytometry experiments.
Disclosures
The authors have no financial conflicts of interest.
References
Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017
1. Batista, F. D., D. Iber, and M. S. Neuberger. 2001. B cells acquire antigen from
target cells after synapse formation. Nature 411: 489–494.
2. Carrasco, Y. R., S. J. Fleire, T. Cameron, M. L. Dustin, and F. D. Batista. 2004.
LFA-1/ICAM-1 interaction lowers the threshold of B cell activation by facilitating B cell adhesion and synapse formation. Immunity 20: 589–599.
3. Dustin, M. L., M. W. Olszowy, A. D. Holdorf, J. Li, S. Bromley, N. Desai,
P. Widder, F. Rosenberger, P. A. van der Merwe, P. M. Allen, and A. S. Shaw.
1998. A novel adaptor protein orchestrates receptor patterning and cytoskeletal
polarity in T-cell contacts. Cell 94: 667–677.
4. Grakoui, A., S. K. Bromley, C. Sumen, M. M. Davis, A. S. Shaw, P. M. Allen,
and M. L. Dustin. 1999. The immunological synapse: a molecular machine
controlling T cell activation. Science 285: 221–227.
5. Krummel, M. F., M. D. Sjaastad, C. Wülfing, and M. M. Davis. 2000. Differential clustering of CD4 and CD3zeta during T cell recognition. Science 289:
1349–1352.
6. Monks, C. R., B. A. Freiberg, H. Kupfer, N. Sciaky, and A. Kupfer. 1998. Threedimensional segregation of supramolecular activation clusters in T cells. Nature
395: 82–86.
7. Potter, T. A., K. Grebe, B. Freiberg, and A. Kupfer. 2001. Formation of supramolecular activation clusters on fresh ex vivo CD8+ T cells after engagement of
the T cell antigen receptor and CD8 by antigen-presenting cells. Proc. Natl.
Acad. Sci. USA 98: 12624–12629.
8. Stinchcombe, J. C., G. Bossi, S. Booth, and G. M. Griffiths. 2001. The immunological synapse of CTL contains a secretory domain and membrane bridges.
Immunity 15: 751–761.
9. Geiger, B., D. Rosen, and G. Berke. 1982. Spatial relationships of microtubuleorganizing centers and the contact area of cytotoxic T lymphocytes and target
cells. J. Cell Biol. 95: 137–143.
10. Kupfer, A., and G. Dennert. 1984. Reorientation of the microtubule-organizing
center and the Golgi apparatus in cloned cytotoxic lymphocytes triggered by
binding to lysable target cells. J. Immunol. 133: 2762–2766.
11. Kupfer, A., S. L. Swain, C. A. Janeway, Jr., and S. J. Singer. 1986. The specific
direct interaction of helper T cells and antigen-presenting B cells. Proc. Natl.
Acad. Sci. USA 83: 6080–6083.
12. Das, V., B. Nal, A. Dujeancourt, M. I. Thoulouze, T. Galli, P. Roux, A. DautryVarsat, and A. Alcover. 2004. Activation-induced polarized recycling targets
T cell antigen receptors to the immunological synapse; involvement of SNARE
complexes. Immunity 20: 577–588.
13. Huse, M., B. F. Lillemeier, M. S. Kuhns, D. S. Chen, and M. M. Davis. 2006.
T cells use two directionally distinct pathways for cytokine secretion. Nat.
Immunol. 7: 247–255.
14. Kupfer, A., T. R. Mosmann, and H. Kupfer. 1991. Polarized expression of
cytokines in cell conjugates of helper T cells and splenic B cells. Proc. Natl.
Acad. Sci. USA 88: 775–779.
15. Reichert, P., R. L. Reinhardt, E. Ingulli, and M. K. Jenkins. 2001. Cutting edge:
in vivo identification of TCR redistribution and polarized IL-2 production by
naive CD4 T cells. J. Immunol. 166: 4278–4281.
16. Iida, T., H. Ohno, C. Nakaseko, M. Sakuma, M. Takeda-Ezaki, H. Arase,
E. Kominami, T. Fujisawa, and T. Saito. 2000. Regulation of cell surface expression of CTLA-4 by secretion of CTLA-4-containing lysosomes upon activation of CD4+ T cells. J. Immunol. 165: 5062–5068.
17. Faroudi, M., C. Utzny, M. Salio, V. Cerundolo, M. Guiraud, S. Müller, and
S. Valitutti. 2003. Lytic versus stimulatory synapse in cytotoxic T lymphocyte/
target cell interaction: manifestation of a dual activation threshold. Proc. Natl.
Acad. Sci. USA 100: 14145–14150.
18. Mitchison, N. A. 2004. T-cell-B-cell cooperation. Nat. Rev. Immunol. 4: 308–
312.
19. Pasare, C., and R. Medzhitov. 2005. Control of B-cell responses by Toll-like
receptors. Nature 438: 364–368.
20. Schnare, M., G. M. Barton, A. C. Holt, K. Takeda, S. Akira, and R. Medzhitov.
2001. Toll-like receptors control activation of adaptive immune responses. Nat.
Immunol. 2: 947–950.
21. Krieg, A. M., G. Hartmann, and A. K. Yi. 2000. Mechanism of action of CpG
DNA. Curr. Top. Microbiol. Immunol. 247: 1–21.
22. Krieg, A. M., A. K. Yi, S. Matson, T. J. Waldschmidt, G. A. Bishop, R. Teasdale,
G. A. Koretzky, and D. M. Klinman. 1995. CpG motifs in bacterial DNA trigger
direct B-cell activation. Nature 374: 546–549.
23. Chaturvedi, A., D. Dorward, and S. K. Pierce. 2008. The B cell receptor governs
the subcellular location of Toll-like receptor 9 leading to hyperresponses to
DNA-containing antigens. Immunity 28: 799–809.
24. Eckl-Dorna, J., and F. D. Batista. 2009. BCR-mediated uptake of antigen linked
to TLR9 ligand stimulates B-cell proliferation and antigen-specific plasma cell
formation. Blood 113: 3969–3977.
25. Depoil, D., R. Zaru, M. Guiraud, A. Chauveau, J. Harriague, G. Bismuth,
C. Utzny, S. Müller, and S. Valitutti. 2005. Immunological synapses are versatile
structures enabling selective T cell polarization. Immunity 22: 185–194.
26. Valitutti, S., and L. Dupré. 2010. Plasticity of immunological synapses. Curr.
Top. Microbiol. Immunol. 340: 209–228.
27. Bertrand, F., M. Esquerré, A. E. Petit, M. Rodrigues, S. Duchez, J. Delon, and
S. Valitutti. 2010. Activation of the ancestral polarity regulator protein kinase C
zeta at the immunological synapse drives polarization of Th cell secretory machinery toward APCs. J. Immunol. 185: 2887–2894.
28. Spörri, R., and C. Reis e Sousa. 2003. Newly activated T cells promote maturation of bystander dendritic cells but not IL-12 production. J. Immunol. 171:
6406–6413.
29. Miro, F., C. Nobile, N. Blanchard, M. Lind, O. Filipe-Santos, C. Fieschi,
A. Chapgier, G. Vogt, L. de Beaucoudrey, D. S. Kumararatne, et al. 2006. T celldependent activation of dendritic cells requires IL-12 and IFN-gamma signaling
in T cells. J. Immunol. 177: 3625–3634.
30. Tourret, M., S. Guégan, K. Chemin, S. Dogniaux, F. Miro, A. Bohineust, and
C. Hivroz. 2010. T cell polarity at the immunological synapse is required for
CD154-dependent IL-12 secretion by dendritic cells. J. Immunol. 185: 6809–
6818.
31. Kupfer, H., C. R. Monks, and A. Kupfer. 1994. Small splenic B cells that bind to
antigen-specific T helper (Th) cells and face the site of cytokine production in
the Th cells selectively proliferate: immunofluorescence microscopic studies of
Th-B antigen-presenting cell interactions. J. Exp. Med. 179: 1507–1515.
32. Kupfer, A., S. L. Swain, and S. J. Singer. 1987. The specific direct interaction of
helper T cells and antigen-presenting B cells. II. Reorientation of the microtubule organizing center and reorganization of the membrane-associated cytoskeleton inside the bound helper T cells. J. Exp. Med. 165: 1565–1580.
33. Kupfer, A., and S. J. Singer. 1989. The specific interaction of helper T cells and
antigen-presenting B cells. IV. Membrane and cytoskeletal reorganizations in the
bound T cell as a function of antigen dose. J. Exp. Med. 170: 1697–1713.
34. Fooksman, D. R., S. Vardhana, G. Vasiliver-Shamis, J. Liese, D. A. Blair,
J. Waite, C. Sacristán, G. D. Victora, A. Zanin-Zhorov, and M. L. Dustin. 2010.
Functional anatomy of T cell activation and synapse formation. Annu. Rev.
Immunol. 28: 79–105.
35. Hiltbold, E. M., N. J. Poloso, and P. A. Roche. 2003. MHC class II-peptide
complexes and APC lipid rafts accumulate at the immunological synapse. J.
Immunol. 170: 1329–1338.
36. de la Fuente, H., M. Mittelbrunn, L. Sánchez-Martı́n, M. Vicente-Manzanares,
A. Lamana, R. Pardi, C. Cabañas, and F. Sánchez-Madrid. 2005. Synaptic
clusters of MHC class II molecules induced on DCs by adhesion moleculemediated initial T-cell scanning. Mol. Biol. Cell 16: 3314–3322.
37. Benvenuti, F., C. Lagaudrière-Gesbert, I. Grandjean, C. Jancic, C. Hivroz,
A. Trautmann, O. Lantz, and S. Amigorena. 2004. Dendritic cell maturation
controls adhesion, synapse formation, and the duration of the interactions with
naive T lymphocytes. J. Immunol. 172: 292–301.
38. Tseng, S. Y., J. C. Waite, M. Liu, S. Vardhana, and M. L. Dustin. 2008. T celldendritic cell immunological synapses contain TCR-dependent CD28-CD80
clusters that recruit protein kinase C theta. J. Immunol. 181: 4852–4863.
39. Pulecio, J., J. Petrovic, F. Prete, G. Chiaruttini, A. M. Lennon-Dumenil,
C. Desdouets, S. Gasman, O. R. Burrone, and F. Benvenuti. 2010. Cdc42mediated MTOC polarization in dendritic cells controls targeted delivery of
cytokines at the immune synapse. J. Exp. Med. 207: 2719–2732.
40. Harris, D. P., L. Haynes, P. C. Sayles, D. K. Duso, S. M. Eaton, N. M. Lepak,
L. L. Johnson, S. L. Swain, and F. E. Lund. 2000. Reciprocal regulation of
polarized cytokine production by effector B and T cells. Nat. Immunol. 1: 475–
482.
41. Lampropoulou, V., K. Hoehlig, T. Roch, P. Neves, E. Calderón Gómez,
C. H. Sweenie, Y. Hao, A. A. Freitas, U. Steinhoff, S. M. Anderton, and
S. Fillatreau. 2008. TLR-activated B cells suppress T cell-mediated autoimmunity. J. Immunol. 180: 4763–4773.
42. Viola, A., R. L. Contento, and B. Molon. 2010. Signaling amplification at the
immunological synapse. Curr. Top. Microbiol. Immunol. 340: 109–122.
43. Yuseff, M. I., D. Lankar, and A. M. Lennon-Duménil. 2009. Dynamics of
membrane trafficking downstream of B and T cell receptor engagement: impact
on immune synapses. Traffic 10: 629–636.
44. Dustin, M. L., and L. B. Dustin. 2001. The immunological relay race: B cells
take antigen by synapse. Nat. Immunol. 2: 480–482.