A distinct Toll-like receptor repertoire in human tonsillar B cells,
directly activated by Pam3CSK4, R-837 and CpG-2006 stimulation
The Toll-like receptor (TLR) family consists of 10 mem-
bers (TLR1–TLR10),1which are transmembrane proteins
with an extracellular leucine-rich domain and a con-
served cytoplasmic domain. The cytoplasmic domain is
homologous to the interleukin (IL)-1 receptor and is
referred to as the Toll/IL-1 receptor (TIR) domain.2–4
Each TLR recognizes specific microbial components, so-
called pathogen-associated molecular patterns (PAMPs),5
including bacterial lipoproteins and lipoteichoic acids
(TLR2), double-stranded viral RNA (dsRNA; TLR3),
Anne Ma ˚nsson, Mikael Adner,
Ulf Ho ¨ckerfelt and Lars-Olaf
Laboratory of Clinical and Experimental
Allergy Research, Department of Otorhino-
laryngology, Malmo ¨ University Hospital,
Lund University, Sweden
Received 24 October 2005; revised 5 April
2006; accepted 5 April 2006.
Correspondence: Anne Ma ˚nsson, Laboratory
of Clinical and Experimental Allergy
Research, Department of
Otorhinolaryngology, Malmo ¨ University
Hospital, Lund University, SE-20502 Malmo ¨,
Sweden. Email: firstname.lastname@example.org
Senior author: Lars Olaf Cardell,
Toll-like receptors (TLRs) recognize specific pathogen-associated mole-
cular patterns (PAMPs), which subsequently trigger innate immunity.
Recent data also suggest a role for TLRs in the direct activation of adap-
tive immune cells. In the present study, the expression and function of
TLR1–TLR10 were characterized in purified human tonsillar B cells,
focusing on differences among CD19+ +CD38– –CD27– –(naı ¨ve B cells),
CD19+ +IgD– –CD27– –[germinal centre (GC) B cells] and CD19+ +CD38– –
CD27+ +(memory B cells) cells. The study was also designed to compare
the TLR expression in B cells obtained from infected and hyperplastic
tonsils that served as controls. The results demonstrated a distinct reper-
toire of TLRs, in which TLR1, TLR2, TLR7, TLR9 and TLR10 predomin-
ated. No differences were found among naı ¨ve, GC and memory B cells.
Tonsillar infection did not substantially alter the TLR expression profile
in ex vivo-isolated B-cell subsets. Purified CD19+ +B cells stimulated with
Pam3CSK4, R-837 and CpG oligodeoxynucleotide (ODN) 2006, via TLR1/
TLR2, TLR7 and TLR9, respectively, showed an induction of interleukin-6
secretion and an up-regulated expression of human leucocyte antigen
(HLA)-DR. Collectively, the present study demonstrates that B cells exhi-
bit constitutively high levels of specific TLRs, which are not developmen-
microbial infections, such as chronic tonsillitis, do not appear to affect
the TLR profile in B cells. Furthermore, the distinct expression of TLRs
allows B cells to respond directly to the cognate PAMPs. This further
emphasizes the role of TLRs in directly activating adaptive immune cells.
Keywords: infection; PAMPs; Toll-like receptors; tonsillar B cells
Abbreviations: Ct, threshold cycle; DAB, 3,30-diaminobenzidine; DC, dendritic cell; dsRNA, double-stranded viral RNA; ELISA,
enzyme-linked immunosorbent assay; FCS, fetal calf serum; FACS, fluorescence-activated cell sorter; FITC, fluorescein
isothiocyanate; GAS, group A b-haemolytic streptococci; GC, germinal centre; HLA, human leucocyte antigen; HRP, horseradish
peroxidase; IFN, interferon; IgD, immunoglobulin D; IgG, immunoglobulin G; IL, interleukin; LPS, lipopolysaccharides; NK,
natural killer; ODN, oligodeoxynucleotide; PAMP, pathogen-associated molecular pattern; PB, peripheral blood; PBL, peripheral
blood lymphocytes; PBMC, peripheral blood mononuclear cell; PBS, phosphate-buffered saline; PDC, plasmacytoid dendritic
cells; PE, phycoerythrin; RT–PCR, reverse transcription–polymerase chain reaction; SRBC, sheep red blood cells; ssRNA, single-
stranded viral RNA; TBS, Tris-buffered saline; TIR, Toll/IL-1 receptor; TLR, Toll-like receptor.
? 2006 Blackwell Publishing Ltd, Immunology, 118, 539–548
IMMUNOLOGY ORIGINAL ARTICLE
lipopolysaccharides (LPS; TLR4), flagellin (TLR5), imida-
zoquinolines and single-stranded viral RNA (ssRNA;
TLR7 and TLR8), and unmethylated CpG oligodeoxynu-
cleotides (ODNs; TLR9).1,4,6–8TLR1 and TLR6 only sig-
nal as a dimer when combined with TLR2,9and the
ligand for TLR10 is as yet unknown.2TLRs are differen-
tially expressed in various immune cells. In general,
mononuclear phagocytes and dendritic cells (DCs) express
the widest TLR repertoires.10Recognition of PAMPs by
innate immune cells results in a signalling pathway that
leads to antigen presentation, up-regulation of costimula-
tory molecules and release of cytokines, which in turn sti-
mulates the adaptive immune system consisting of B and
T lymphocytes.9–11In addition to this indirect activa-
tion of B cells, it is also speculated whether infectious
organisms can directly activate the adaptive immune
At present, our knowledge about TLR regulation and
function in B cells is limited, except for TLR9. Herein, we
examined whether the expression of TLRs is developmen-
tally regulated during the differentiation from naı ¨ve to
germinal centre (GC) and memory B cells in tonsillar tis-
sue, and whether the TLR expression in the various cell
subsets is regulated in response to infection. The direct
effects of TLR ligands other than CpG have been poorly
investigated on purified human B cells. Therefore, we also
analysed the functional response of purified human B
cells to a range of different TLR ligands.
Materials and methods
Tonsils were obtained from 33 patients undergoing ton-
sillectomy at Malmo ¨ University Hospital (Malmo ¨, Swe-
den). The study was approved by the Ethics Committee
of Lund University and written informed consent was
obtained. After the tonsillectomy, swabs were taken for
tonsillar core cultures (representing the microbial flora of
the tonsillar crypts) in order to determine the presence
of pathogenic b-haemolytic streptococci and anaerobes.
The patients providing the tonsils were divided into two
groups (referred to as infected or control), based on their
clinical diagnoses and the outcome of the core culture.
The infected group consisted of 14 patients (age range:
3–21 years, median 7 years) referred for tonsillectomy
because of multiple episodes (at least four times during
the year preceding the surgery) of group A b-haemolytic
enlarged tonsils, positive culture test). The control group
consisted of 19 patients (age range: 3–18 years, median
6?5 years) who underwent tonsillectomy because of tonsil-
lar hyperplasia indicated by either obstructive sleep
apnoea or occasional failure to thrive (no history of
recurrent tonsillitis, negative culture test). Even though
the control tonsils were culture-negative and functioned
as appropriate controls in regard to infection, they were
not normal healthy tissues. Therefore, it cannot be
entirely excluded that there was some immunologically
relevant cause of the hyperplasia that affected the out-
come of the present study. None of the patients displayed
symptoms of acute infection at the time of surgery, and
none had received any antibiotic treatment for at least
1 month before surgery. Apart from the tonsillar symp-
toms, all patients were healthy and did not receive any
Antibodies and reagents
The following anti-human immunoglobulins were used
for flow cytometry analyses: CD19-ECD (J4.119) from
Immunotech (Beckman Coulter, Marseille, France), CD27-
phycoerythrin (PE) (M-T271) from DakoCytomation
(Copenhagen, Denmark), CD38-fluorescein isothiocyanate
(FITC) (HIT2) from BD Pharmingen (Heidelberg, Ger-
many), immunoglobulin D (IgD)-FITC (goat, polyclonal),
TLR2-FITC (TL2.1) and TLR9-FITC (26C593) from AMS
Biotechnology (Abingdon, UK) and human leucocyte anti-
gen (HLA)-DR-PE (LN3) from eBioscience (San Diego,
CA). Unlabelled antibodies against TLR1 (GD2.F4) and
TLR7 (rabbit, polyclonal) were purchased from Acris anti-
bodies (Hiddenhausen, Germany), and detected using the
Alexa Fluor 488 mouse immunoglobulin G1 (IgG1) label-
ling kit from Molecular Probes (Eugene, OR) or sheep
anti-rabbit IgG-PE from Acris. For immunohistochemical
staining, antibodies against CD3 (PSI) from Novocastra
(Newcastle upon Tyne, UK), CD20cy (L26) from Dako-
Cytomation, TLR1 (GD2.F4), TLR2 (rabbit, polyclonal),
TLR7 (rabbit, polyclonal) and TLR9 (26C593) from Acris
were used. For visualization, DakoCytomation Envision+
System-horseradish peroxidase (HRP) kits were utilized.
Pam3CSK4, flagellin (from Bacillus subtilis) and imiquimod
(R-837) were purchased from Invivogen (San Diego, CA),
LPS (from Salmonella minnesota) from Alexis Biochemi-
cals (Lausen, Switzerland) and phosphorothioate-modified
CpG ODN 2006 (CpG-2006), 50-tcgtcgttttgtcgttttgtcgtt-3013
from DNA Technology A/S (Aarhus, Denmark).
Cell separation and cell culture
Tonsils were minced in complete medium consisting of
RPMI-1640 (Sigma-Aldrich, St Louis, MO) supplemented
with 0?3 g/l
(AH diagnostics, Aarhus, Denmark), 100 U/ml penicillin
and 100 lg/ml streptomycin (Invitrogen, Carlsbad, CA),
and the T cells were removed by rosetting with neur-
Mononuclear T-cell-depleted cells were isolated by den-
sity-gradient centrifugation using Ficoll–Paque (Amer-
L-glutamine, 10% fetal calf serum (FCS)
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