Glycosylation of surface Ig creates a functional bridge
between human follicular lymphoma and
Vania Coelhoa,1, Sergey Krysova, Amir M. Ghaemmaghamib, Mohamed Emarab, Kathleen N. Pottera, Peter Johnsona,
Graham Packhama, Luisa Martinez-Pomaresb, and Freda K. Stevensona,1
aMolecular Immunology Group, Cancer Sciences Division, University of Southampton, School of Medicine, Southampton SO16 6YD, United Kingdom; and
bSchool of Molecular Medical Sciences, Institute of Infection, Immunity and Inflammation, Queen’s Medical Centre, University of Nottingham, Nottingham
NG7 2UH, United Kingdom
Edited* by Ronald Levy, Stanford University, Stanford, CA, and approved September 22, 2010 (received for review July 1, 2010)
Surface Ig (sIg) of follicular lymphoma (FL) is vital for tumor cell
survival. We found previously that the Ig in FL is unusual, because
the variable region genes carry sequence motifs for N-glycan ad-
dition. These are introduced by somatic mutation and are tumor
specific. Unexpectedly, added glycans terminate at high mannose,
suggesting a potentially important interaction of FL cells with
mannose-binding lectins of the innate immune system. We have
now identified mannosylated IgM at the surface of primary lym-
phoma cells. Recombinant lectin domains of the mannose receptor
(MR) or DC-SIGN bind mannosylated Igs in vitro and bind to FL
cells, signaling sIgM-associated increases in intracellular Ca2+. Lec-
tins also bind to normal B cells but fail to signal. In contrast, anti-Ig
signaled similarly in both FL and normal B cells. Mannosylation
patterns were mimicked by FL Ig-derived single-chain Fvs (scFv),
providing probes for potential receptors. Mannosylated scFv
bound specifically to the lectin domains of the MR and DC-SIGN
and blocked signaling. Mannosylated scFv also bound to DC-SIGN
on the surface of dendritic cells. This unique lymphoma-specific
interaction of sIg with lectins of innate immunity reveals a poten-
tial route for microenvironmental support of tumor cells, mediated
via the key B-cell receptor.
B cell|B-cell receptor|B-cell lymphoma|immunoglobulin
pression of the BCL-2 oncoprotein (1). Although the trans-
location disrupts one Ig allele, expression of surface Ig (sIg) is
retained, suggesting a role for sIg in tumor cell behavior. In-
terestingly, normal subjects can have B cells with the charac-
teristic translocation (2), and cells with features of “precursor”
cells have been detected, especially after exposure to environ-
mental toxins (3). These observations suggest a multistep route
One potential step was revealed by our finding that the vari-
able region genes of the Ig in FL have accumulated acceptor
sequence motifs for addition of N-glycans (4). These sequence
motifs are introduced during somatic hypermutation and are
characteristic only of FL and some germinal center tumors, being
very infrequent in normal B cells, in chronic lymphocytic leu-
kemia, or in myeloma (5). The frequency of motifs in FL-derived
IGHV sequences deposited in the database was 55 of 70 (79%),
as compared with 7 of 75 (9%) in normal somatically mutated B
cells (4). This high incidence was confirmed in a study of FL
cases entered into a clinical trial, in which 24 of 24 cases had
motifs (6). Because both studies were confined to IGHV, but it is
now known that motifs can be found in IGLV (7), this indicates
that the overall incidence in FL may approach 100%.
Another finding was that the glycans added to the motifs were
unusual in terminating at high mannose, a feature generally con-
fined to “immature” glycoproteins in the endoplasmic reticulum.
Most surface glycoproteins, including the constant regions of
ollicular lymphoma (FL) generally develops from B cells that
have undergone the t(14;18) translocation, up-regulating ex-
proteins is therefore unexpected, and the fact that mannoses were
located in the variable regions of the sIg strongly suggested that
they could be influencing the behavior of FL cells. Our hypothesis
(9)was thatthere wasan opportunistic interaction between the B-
cell receptor of FL cells with a mannose-binding lectin in the
is evidence for constitutive activation of FL cells in vivo (10).
The observations that genes primarily associated with mono-
cytesand dendritic cells (DCs) arepredictive ofa poorer outcome
in FL (11), and that higher numbers of CD68+macrophages are
associated with a poorer prognosis (12), reveal candidate cells for
this interaction. Importantly, macrophages located both within
and between the follicles seem to be predictors of disease pro-
gression (13). The activation status of tumor-associated macro-
phages may also be critical, with Stat-1 expression, essential for
IFNγ signaling, an apparently adverse prognostic factor (14).
Although prognosis is complicated by combining pathogenesis
with response to therapy, these findings place FL-associated
macrophages in line with other tumor-associated macrophages
that have been shown to promote tumor progression (15) by
multiple pathways, possibly including suppression of immune
Candidate molecules for interaction with mannosylated Ig in-
clude C-type lectins that bind carbohydrate structures in a Ca2+-
dependent manner. C-type lectins act as pattern recognition
receptors (17), able to bind to pathogens and activate immunity
(18). They also bind to endogenous ligands and mediate cell–cell
interactions during immune responses (18). Two major C-type
lectins with specificity for high mannose structures are the man-
nose receptor (MR) and DC-SIGN (DC-specific intercellular
adhesion molecule-3–grabbing nonintegrin) expressed by cells of
innate immunity, including macrophages and DCs (19).
We have now demonstrated that binding of mannose-binding
lectins to FL triggers B-cell receptor–mediated signaling events,
thereby establishing a functional connection between primary
lymphoma cells and the innate immune system. Conversely,
mannosylated Ig derivatives can bind to specific lectins on DCs.
This functional bridge to the key B-cell receptor in FL could
Author contributions: V.C., L.M.-P., and F.K.S. designed research; V.C., S.K., and M.E.
performed research; A.M.G. contributed new reagents/analytic tools; V.C., S.K., K.N.P.,
The authors declare no conflict of interest.
*This Direct Submission article had a prearranged editor.
1To whom correspondence may be addressed. E-mail: firstname.lastname@example.org or vcds1b06@soton.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| October 26, 2010
| vol. 107
| no. 43
replace the need for antigen to promote survival of B cells in the
Surface IgM of Primary Lymphoma Cells Expresses Mannosylated IgM.
The first question was whether primary lymphoma cells expressed
mannosylated IgM at the cell surface. We therefore investigated
primary lymphoma cells directly, randomly selecting two FL cases
known to have mannosylated N-glycosylation motifs in the Ig
sIgM, and this was exposed to specific glycosidases, Endo-H and
peptide:N-glycosidase (PNGase), which cleave either mannosy-
lated or all N-glycans, respectively. Although both FL and normal
B cells carry mature PNGase-cleavable glycans at sites in the
constant regions, the pattern of Endo-H cleavage of the bio-
tinylated sIgM shows clearly that the FL cases, but not normal B
cells, carry additional mannosylated glycans (Fig. 1). Endo-H
cleavage of mannosylated glycans of sIgM increased mobility on
the gel in both cases, with a greater effect in case FL21 as com-
samples and from normal B cells, leading to μ chains of ≈70 kDa.
the migration distance after treatment as a proportion of the
therefore that the mannosylation of the Ig variable regions of FL
cells occurs in vivo. This validates the findings of mannosylated
sites in lymphoma-derived Ig expressed by heterohybridization,
analysis of which had located mannosylation to the Ig variable
human cells provides a faithful reproduction of the natural gly-
cosylation pattern in vitro. Using this expression system, we
detectedmannosylation in sevenofseven randomlyselectedcases
of FL (7).
Mannosylated Ig and scFv Bind to C-Type Lectins in Vitro. To assess
the potential functional importance of the mannosylated sIg, we
cases of FL to bind to recombinant lectins. As controls we used
nonmannosylated scFvs, or purified whole monoclonal IgG, de-
glycosylation sites in the variable regions (7). The MR consists of
multiple functional domains, and these were expressed either as
the C-type lectin-like domains (CTLD) 4–7, comprising the man-
nose-binding domain (CTDL 4), or, as a control, CR-FNII-CTLD
1–3, comprising the cysteine-rich domain, the fibronectin type II
domain, and CTLDs 1–3 (Fig. S1). These were designated as
Lectin-MR and Control-MR, respectively. DC-SIGN is a type II
transmembrane protein. The extracellular domain of this lectin
a C-type carbohydrate recognition domain. The recombinant DC-
SIGN-Fc available was expressed as the extracellular portion
(amino acid residues 64–404). Each recombinant lectin is fused to
human Fcγ for detection and purification. Using scFv-Cκ mole-
cules derived from the two cases of FL above, together with seven
further randomly selected cases with one to five glycosylation sites
MR(Fig.S2i).In theabsence ofany lectintherewasno detectable
binding signal; therefore, the very low level of binding of the
Control-MR indicates a minor interactive ability with the scFv
molecules, more evident in the MM-derived nonmannosylated
scFvs, where there was no significant specific lectin binding (Fig.
S2i). There was variability in the relative binding capacity of the
various mannosylated scFv molecules for Lectin-MR and DC-
SIGN. A similar variable but specific pattern was obtained using
whole IgG derived from three additional FL cases by hetero-
hybridization, with no specific binding of a control IgG myeloma-
derived protein (Fig. S2ii). These data confirm the biochemical
data (7, 9) showing that FL-derived Ig from a range of patients,
expressed in two molecular forms, and in different mammalian
expression systems, are mannosylated, and reveal that mannoses
are exposed in a configuration that allows binding to the lectins.
C-type Lectins Bind to FL Cells and to Normal B Cells. The ability of
the lectins to bind to primary FL cells from two patients was then
investigated by FACS. Both Lectin-MR and DC-SIGN showed
strong Ca2+-dependent binding to FL cells, with considerably less
domain, which has specificity for sulfated glycans (20).
Surprisingly, Ca2+-dependent binding by Lectin-MR and DC-
SIGN also occurred to normal human B cells, which do not carry
mannosylated sites in sIgM (Fig. 2C). There was some binding of
the Control-MR to normal B cells, which seemed to be largely
Ca2+independent. The pattern of lectin and control binding to
normal B cells was similar to FL cells, except that mean fluo-
rescence intensity (MFI) for both lectins was lower than for the
FL cases. For FL, the major question was whether the outcome
FL patients with different numbers of VH-region N-glycosylation (GLY) sites.
Viable normal B cells were purified by negative selection from PBMCs. Cell-
surface proteins were isolated from each sample and treated with Endo H or
analyzed by immunoblotting using an anti-μ antibody. Reproducibility was
assessed by repeating the immunoblotting three times with similar results.
Surface IgM-derived μ chains of primary FL B cells carry mannosylated
bindingbyliveCD19+B cellsfromtwoFLcases (AandB)andfromonehealthy
individual (C) was analyzed by FACS. Shaded lines: secondary antibody (anti-
binding intheabsenceofCa2+.Bindingofeachindividual lectintoFLcellswas
reproduced twice for each sample. Binding to normal B cells is representative
of the same results obtained with B cells from three different donors.
| www.pnas.org/cgi/doi/10.1073/pnas.1009388107 Coelho et al.
of binding differed from that of normal B cells in terms of sIgM-
mediated intracellular signals.
C-Type Lectins Mediate Intracellular Signaling Only in FL. Surface
IgM is a key component of the B-cell receptor and mediates
intracellular signals important for proliferation or apoptosis (21).
We found that polyclonal F(ab′)2anti-μ antibody led to a Ca2+
flux in FL29 cells, as expected (Fig. 3 Ai and Aii). However, the
striking finding was that lectins were also able to mediate signals
in FL cells, with Lectin-MR (Fig. 3Ai) and DC-SIGN (Fig. 3Aii)
each generating a Ca2+flux. For both lectins, the signal was
weaker and slower than that induced by anti-μ, possibly owing to
binding of the polyclonal anti-μ to multiple sites on sIgM.
However, the response to Lectin-MR could be amplified by prior
cross-linkage using anti-Fcγ (Fig. 3Ai), with higher numbers of
responding cells and a faster response. The effect of cross-link-
age on DC-SIGN was less evident, with only a modest increase in
rate of response observed (Fig. 3Aii). This lesser dependence of
signaling efficacy on cross-linkage via Fcγ could reflect the nat-
ural ability of DC-SIGN to form tetramers via the neck region of
the molecule (22). No effect was seen using the cross-linked
Control-MR (Fig. 3Ai). It seems that, although the lectin
domains can presumably dimerize via the fused human Fcγ
portion, multimerization increases the rate and level of the
specific signal, at least for Lectin-MR. Confirmation of the ability
of cross-linked Lectin-MR and DC-SIGN to induce a Ca2+sig-
nal in FL cells was obtained using FL21 (Fig. 3Aiii) and was
confirmed in four of four additional FL cases (Fig. S3).
For Lectin-MR, signaling in FL29 was completely blocked by
preincubation with the homologous FL-derived mannosylated
scFv at a molar ratio of scFv:Lectin-MR of 2:1, but not by
nonmannosylated scFv (Fig. 3Bi), indicating that, for this lectin,
Ig-derived mannosylated scFv was a strong competitor for the
cell-surface target. DC-SIGN–induced signaling was consider-
ably but not totally blocked by mannosylated scFv (Fig. 3Bii) at
a molar ratio of scFv:DC-SIGN of 7:1, and inhibition could not
be increased significantly by raising this to 15:1. The requirement
for higher levels of scFv for blocking could again reflect the
multimeric nature of the recombinant DC-SIGN, which would
confer a high avidity for the cell surface IgM. An alternative
possibility is that DC-SIGN may be capable of mediating an
additional weaker signal in FL cells, possibly unrelated to its
lectin domain. Although ligands for the non-CTLD domains of
DC-SIGN are known (23), most investigators have studied back
signaling into the DC rather than into a target cell; therefore,
this observation needs further investigation.
point shown by the arrow. B cells were gated using anti-CD19. The y-axis represents percentages of cells over the threshold, set at the 85th percentile of
unstimulated CD19+cells. (A) FL29 cells exposed to anti-μ or to lectins, (i) MR, or (ii) DC-SIGN with or without cross-linkage (X). (iii) FL21 exposed to anti-μ or to
X-Lectins. (B) Blocking of lectin stimulation of FL29 cells by preincubation of (i) Lectin-MR or (ii) DC-SIGN with mannosylated (Mann) scFv [or control non-
mannosylated (Non-Mann)scFv]atamolarratioof1:2(MR)or1:7(DC-SIGN).(C)NormalBcellspurifiedfrom(i)subject 1exposedtoanti-μortoX-LectinMR;(ii)
subject 2 exposed to anti-μ or to X-DC-SIGN; (iii) PBMCs from subject 3 exposed to anti-μ or to X-Lectin-MR or to X-DC-SIGN. In all cases, anti-μ was F(ab)2.
Coelho et al. PNAS
| October 26, 2010
| vol. 107
| no. 43
For normal blood B cells from two subjects (1 and 2), the
response to anti-μ was strong and similar to that in the FL cells
(Fig. 3 Ci and Cii). The effect of cross-linked lectins on normal B
cells was then investigated, using the same conditions as for FL
cells. No lectin-mediated signaling was detectable in normal B
cells, with cross-linked Lectin-MR failing to generate any de-
tectable signal (Fig. 3Ci). The second normal donor was tested
with cross-linked DC-SIGN, which also was completely negative
(Fig. 3Cii). This donor was found to have 41.8% of CD27-posi-
tive B cells within the CD19-positive population, therefore the
failure to signal indicates that neither naïve nor memory B cells
responded to Lectin-MR. A further normal donor was tested
using unselected peripheral blood mononuclear cells (PBMCs),
locating B cells by FACS with anti-CD19. Although B-cell
numbers were low, again a response to anti-μ and no response to
either lectin were observed (Fig. 3Ciii). The discriminatory out-
come indicates that lectin interaction with mannosylated sites
can distinguish between FL cells and normal cells, whereas anti-μ
cannot. Evidently, binding to the non-Ig receptors on the surface
of normal B cells (and presumably on FL cells) occurs (Fig. 2),
but only the binding to the mannosylated sIgM and not to
nonmannosylated sIgM or to other cell-surface molecules, leads
To directly locate the binding of the lectins to the sIgM of the
FL cells, we investigated the ability of the bound cross-linked
lectins to inhibit subsequent binding of anti-μ. For FL29, the
expression of sIgM indicated two populations (Fig. 4Ai), but
staining for BCL-2 was positive for both; therefore, all of the
sIgM-positive cells seem to be FL cells. Blocking was evident and
equivalent using either Lectin-MR or DC-SIGN (Fig. 4Ai). For
FL21, where sIgM expression was again heterogeneous, blocking
was seen with both lectins but seemed more efficient with DC-
SIGN (Fig. 4Aii). In both cases, no blocking was detected using
Control-MR. To ensure that the cross-linked lectins bound to
the cell surface were not inhibiting access of antibodies against
other cell-surface molecules nonspecifically, the effect of lectins
on binding of anti-CD19 was assessed. No effect was observed
(Fig. 4 Aiii and Aiv) for either FL case. The ability of the lectins
to at least partially block binding of the polyclonal anti-μ to
sIgM, even though the binding sites differ, locates lectin binding
The next question concerned the effect of lectins on binding of
anti-μ to normal B cells. Normal sIgM-positive B cells in blood
comprise naïve and memory B cells, with the latter expressing
higher levels of sIgM (24, 25). No effect of lectin exposure on the
access of anti-μ to the total IgM+B-cell population in blood was
detected (Fig. 4Bi). Because FL cells resemble memory B cells
more closely than naïve B cells, we also specifically investigated
CD27+memory B cells. These cells express more sIgM, but again
no lectin-mediated blocking of anti-μ was detectable (Fig. 4Bii). It
seems that lectins bound to sIgM in FL cases are able to block
access of anti-μ, but lectins bound to other cell-surface molecules
there is FL-specific lectin-mediated signaling via sIgM.
Dendritic Cells Bind to Mannosylated scFv via Specific Receptors. To
determine whether cell surface–expressed lectins can recognize
mannosylated Ig, mannosylated scFv probes derived from FL or
nonmannosylated scFvs were used. Dendritic cells were gener-
ated from monocytes in vitro. Mannosylated scFvs from two
patients showed Ca2+-dependent binding to DC, with no binding
of nonmannosylated scFvs (Fig. 5A). Knockdown of DC-SIGN
expression by the DCs was achieved using specific siRNA, shown
by reduction (69%) of mRNA levels, with no effect on mRNA
from the MR (Fig. S4i). Protein expression of DC-SIGN was
partially reduced, again without affecting expression of MR (Fig.
S4ii). By gating on the knocked-down population of DCs (R1
gate, Fig. S4ii), it was possible to analyze the level of binding of
mannosylated scFvs to DC-SIGN.
For both FL cases, binding of the derived mannosylated scFv
in DCs with reduced DC-SIGN expression was less than that to
cells treated with control siRNA (Fig. 5 Bi and Bii). However,
some binding ability remained, possibly due to MR or to other
receptors. It was not possible to knock down MR specifically
using currently available siRNA. The conclusion is therefore that
mannosylated scFv clearly binds to DC-SIGN on DCs, but the
question of involvement of other receptors remains open.
Development of FL is known to require more than the t(14;18)
translocation, assumed to have occurred in the bone marrow. We
propose that another step in the evolution of lymphoma occurs
as the prelymphoma cell differentiates and involves the acqui-
sition of N-glycosylation sites during somatic mutation (Fig. S5).
Retention of the introduced glycan addition sites by FL cells over
time of disease supports this concept (7). Added sugars are un-
usual in terminating at high mannose, normally a biosynthetic
precursor glycoform in the endoplasmic reticulum.
The previous finding that whole Ig expressed from FL cells by
but has fully N-glycosylated sites in the constant region (9) indi-
cates steric influences on accessibility of the IgM domains to gly-
cosyltransferases in the Golgi stacks. This is perhaps more likely
than an influence of adjacent primary V-gene sequences, espe-
cells from two patients, FL29 (i) and FL21 (ii), were exposed to Lectin-MR, to DC-
from a normal subject were exposed to lectins and then to anti-μ as above. (i) B
by memory B cells. Reproducibility was assessed by repeating the blocking assay
the IGHV and IGLV genes of each case is indicated.
| www.pnas.org/cgi/doi/10.1073/pnas.1009388107 Coelho et al.
mutational changes. One study of an unusual mouse monoclonal
anti-dextran antibody reported addition of complex mature gly-
cans at a natural site in the CDR2 of VH but mannosylation at an
introduced site nearby. Importantly the same pattern of glycosyl-
ation occurred using several expression systems, indicating an in-
trinsic structural feature (26). Our studies, using two expression
systems and two molecular forms of FL-derived Ig, support this
consistency, with glycan composition verified by susceptibility to
Endo-H enzyme and by sugar analysis (7).
However, it was important to identify mannosylated sIgM on
primary FL cells, and this is now clear. Its functional role is in-
dicated by identifying a unique lectin-mediated signaling path-
way operating on the FL B-cell receptor but not on normal B
cells. This discriminatory signal could release the transformed B
cells from dependence on antigen for growth or survival in the
hostile environment of the germinal center.
There is a forest of glycoproteins expressed at cell surfaces,
with different N-glycan patterns, although termination at high
mannose is rare. Glycans have many functions, including the
cell–cell interactions required for adhesion and migration (18).
Clearly a wide range of lectins can bind to multiple glycans
expressed on the cell surface of both FL and of normal B cells.
Binding of C-type lectins to normal human B cells has been
reported previously using mannose-binding lectin as a probe
(27), and binding of DC-SIGN is apparently increased in cells of
B-lineage acute lymphoblastic leukemia (28). However, lectins
are rarely completely specific for particular sugars, and it is un-
clear which oligosaccharides were responsible for lectin binding
to these B cells or what the functional significance is.
Certainly, binding per se does not mediate signaling to the B
cell, as measured by Ca2+flux. It seems that only if the lectin
binds specifically to the mannosylated sIg of the B-cell receptor
can a signal be generated. This is in contrast to the comparable
signal detected in both normal and neoplastic B cells when sIg is
engaged by anti-μ. A previous study of FL reported that anti-Ig
induced phosphorylation of Btk, Syk, and p38 even more effec-
tively than in the infiltrating nontumor B cells (29). We did not
observe a weaker signaling in normal B cells compared with FL
cells, and this differential may be confined to intratumoral nor-
mal B cells (29). The critical point is that whereas both FL and
normal B cells, of naïve or memory type, can respond to en-
gagement of sIg with anti-Ig, only FL cells, which bear mannoses
in the variable region of the Ig, respond to lectins. However,
normal B cells are highly heterogeneous, and it would be
expected that motifs would be generated at some level during
somatic mutation. Analysis of centroblasts/centrocytes in a re-
active lymph node would be useful to investigate this point. The
question is whether antigen selection would act against accu-
mulation of these cells in the memory pool. A small percentage
of B cells clearly acquires N-glycosylation sites in IGHV genes,
and in an unusual example, the presence of glycan increased the
affinity of a mouse monoclonal antibody for a carbohydrate
dextran antigen (30). However, few motifs are found in the hu-
man memory B-cell population, indicating that, in contrast to FL
cells, this feature is rarely positively selected.
The nature of the partner lectin-expressing cell is not yet
known, and identification will require exploration of cell–cell
interactions in vitro and detailed probing of candidates in FL
tissues. Among the candidate lectin-expressing cells in FL are
macrophages, known to be associated with a shorter survival (12,
31), and DCs, implicated in prognosis by gene expression pro-
filing (32). Both express lectins able to signal FL cells, potentially
creating a functional bridge between the key sIg receptor,
retained and mannosylated in the vast majority of FL cases, and
a microenvironmental cell-bound lectin.
In terms of lymphomagenesis, a multistep pathway is likely,
with the t(14;18) translocation as the first step. It seems that
transformed cells can respond to antigen and establish a germi-
nal center (33), where somatic mutation, documented to occur
after transformation from intraclonal variation in FL V-gene
sequences, takes place (Fig. S5). Acquisition of N-glycans would
be another step in lymphomagenesis, because tumor cells
retained in the hostile germinal center, with dwindling antigen
levels, could obtain antigen-independent support via interaction
of mannosylated sIg with adjacent lectins. This opportunistic
strategy may exploit the microenvironment for growth and sur-
vival of tumor cells (Fig. S5). Clearly cell–cell interactions must
be analyzed, but we are concerned that FL-derived cell lines may
not retain glycosylation patterns characteristic of cells in vivo,
making primary lymphoma the only feasible target for in-
vestigation and therefore limiting the investigational scope.
The clinical relevance is that interruption of this interaction
could be beneficial for patients, even though the partner cells
involved remain unknown. It is possible that the efficacy of anti-
idiotypic antibody (34) depended at least in part on blocking this
vital interaction. However, rather than individual antiidiotypic
antibodies being required, an antibody or small molecule capable
of blocking a mannose–lectin interaction could provide a single
therapy for FL targeting the same critical molecule, sIg.
Materials and Methods
Patient and Healthy Donor Material. Samples of primary cells from lymph
nodes of two FL patients and PBMCs from healthy donors were isolated from
frozen stocks (35). Approval was obtained from the Southampton and South
West Hampshire Research Ethics Committee. Informed consent was provided
in accordance with the Declaration of Helsinki.
Biotinylation and Glycosylation Analysis of Cell Surface Proteins. Surface pro-
teins of cells (>90% viability) were labeled with biotin (35). Analysis of gly-
cells via DC-SIGN. Dendritic cells were prepared from blood monocytes and
tested for binding of mannosylated scFv-Cκ or nonmannosylated (Non-
Mann) scFv-Cκ by FACS using anti-Cκ| for detection. (A) Binding of man-
nosylated scFv was specific and Ca2+dependent. (B) Dendritic cells with
minimal levels of DC-SIGN after knockdown (R1 gate in Fig. S4ii) were tested
for binding of mannosylated scFv from two FL patients by FACS. Result
shown is representative of three independent experiments. Shaded lines:
anti-Cκ| alone; heavy black lines: binding to knocked down (R1) cells; dashed
lines: binding to dendritic cells treated with control siRNA.
FL-derived mannosylated (Mann) scFv binds to immature dendritic
Coelho et al.PNAS
| October 26, 2010
| vol. 107
| no. 43
cosylation patterns was performed by assessing susceptibility of the isolated
surface proteins to Endo-H and PNgase (35).
Recombinant Proteins. FL-derived scFv-Cκ proteins were assembled and
expressed (7). FL-derived complete Igs were generated via hetero-
hybridomas (9). Fc-chimeric proteins containing different regions of the
mannose receptor (CR-FNII-CTDL1-3-Fc and CTDL4-7-Fc) were generated (36,
37). DC-SIGN-Fc was purchased from R&D Systems. Recombinant lectins were
cross-linked by 30-min incubation with a mouse monoclonal anti-human IgG,
γ chain-specific (Southern Biotech) at a molar ratio of ≈1:1, at 4 °C before
Analysis of Lectin Binding to Cells by Flow Cytometry. FL B cells or normal B
cells were thawed, washed, and allowed to recover in medium for 1 h (35).
Single-cell suspensions were left on ice in lectin buffer [10 mM Tris (pH 7.4),
154 mM NaCl, 3% BSA, 2 mM NaN3,supplemented or not with 10 mM CaCl2]
for 45 min. Binding of recombinant lectins to the B cells was analyzed by
adding lectin constructs (with or without cross-linkage) at saturating levels
of 20 μg/mL and detected by a polyclonal anti-human Fcγ (Jackson Immu-
noResearch) at 10 μg/mL. Simultaneously, B cells were also stained with anti-
CD19APC or anti-CD27APC (both from Biolegend).
For analysis of binding to monocyte-derived DCs, differentiated cells were
harvested from culture on day 6. Binding of the mannosylated and non-
mannosylatedscFvs tothe DCs was tested by adding the scFvs at 15 μg/mL and
detecting by an anti-Cκ light chain phycoerythrin antibody (Dako). All
incubations and washing steps were performed in lectin buffer as above.
Measurement of Intracellular Ca2+. Calcium mobilization was measured using
the fluorogenic probe Fluo 3-AM (38). Cells were also stained with anti-CD19
APC labeled (Biolegend). Cells were stimulated with 10 μg/mL of either anti-μ
or recombinant lectins and FL1 recorded over the next 5 min. To test
blocking of lectin binding by scFvs, recombinant lectins were incubated with
scFvs, at a molar ratio of scFv:DC-SIGN of 7:1 and scFv:Lectin-MR of 2:1, at
4 °C for 30 min before stimulation. For these assays the DC-SIGN concen-
tration was reduced to 5 μg/mL. Data were analyzed using FlowJo software.
Monocyte Differentiation and DC-SIGN Knockdown. To determine whether cell
surface lectins expressed by immune cells can recognize mannosylated Ig, FL-
or MM-derived scFvs were used as tools. Immature DCs were generated from
CD14+monocytes obtained from healthy donors (39). DC-SIGN knockdown
was performed on day 1 of differentiation. Transfection mix, added to each
well seeded with 8 × 105cells, consisted of 3 μL of HiperFect transfection
reagent (Qiagen; ref. no. 301705) and DC-SIGN–specific siRNA (oligo ID
HSS121249; Invitrogen) or control siRNA (Qiagen) at a final concentration of
100 nM, in 100 μL of RPMI without serum. Transfection mix was initially
incubated at room temperature for 15 min before being added to each well
containing cells in another 100 μL of culture media. Transfection was initially
carried out for 4 h at 37 °C, 5% CO2, in a 24-well plate. After this period 600
μL of supplemented media was added to each well, and cells were allowed
to differentiate in the transfecting media for another 5 d. By day 6, differ-
entiated monocytes were harvested for analysis. Monitoring of the level of
expression of MR and DC-SIGN was performed by staining cells with anti-
CD206 or anti-CD209 (Beckman Coulter), respectively.
Estimation of DC-SIGN Receptor Knockdown by Quantitative PCR. cDNA ex-
traction protocol and primer sequences are described in SI Materials
ACKNOWLEDGMENTS. We thank Dr. Dan Mitchell for discussion; Human
Tissue Bank technicians Anna Tilbury and Jessica Borras for help; Dr. Katy
McCann (Cancer Sciences Division, University of Southampton) for providing
the FL-derived scFvs and for advice; and Dr. Maurizio Bendandi (Center for
Applied Medical Research, University ofNavarra) for providing theFL-derived
Igs. This work was supported by Cancer Research UK, the Kay Kendall Leukae-
mia Fund, Asthma UK, and the Southampton Department of Health/Cancer
Research UK Experimental Cancer Medicine Centre.
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