A case of severe autoimmune haemolytic anaemia due to clinically significant ...Apollo Hospitals
Anti N antibody belongs to the MNS blood group system. Usually, anti N antibodies are naturally occurring, cold agglutinins. Clinically significant anti N antibodies have also been reported. We report here a case of autoantibody with anti N specificity presenting with severe autoimmune haemolytic anaemia.
T cell recall response of two hypothetical proteins (Rv2251 and Rv2721c) from...Santhi Devasundaram
The demonstrated variable efficacy of the only licensed TB vaccine Mycobacterium
bovis bacillus CalmetteeGue´rin (M. bovis BCG) encourages the need for new vaccine candidates
against TB. Antigen specific cellular immune response is often considered imperative
during Mycobacterium tuberculosis (M. tuberculosis) infection and antigens that are strongly
associated with the latent phase of infection are drawing increasing attention for anti-TB vaccine
development. Here, we investigated the phenotypic and functional profiles of two novel
mycobacterial antigens Rv2251 and Rv2721c during T cell recall response via multi-color flow
cytometry.
A case of severe autoimmune haemolytic anaemia due to clinically significant ...Apollo Hospitals
Anti N antibody belongs to the MNS blood group system. Usually, anti N antibodies are naturally occurring, cold agglutinins. Clinically significant anti N antibodies have also been reported. We report here a case of autoantibody with anti N specificity presenting with severe autoimmune haemolytic anaemia.
T cell recall response of two hypothetical proteins (Rv2251 and Rv2721c) from...Santhi Devasundaram
The demonstrated variable efficacy of the only licensed TB vaccine Mycobacterium
bovis bacillus CalmetteeGue´rin (M. bovis BCG) encourages the need for new vaccine candidates
against TB. Antigen specific cellular immune response is often considered imperative
during Mycobacterium tuberculosis (M. tuberculosis) infection and antigens that are strongly
associated with the latent phase of infection are drawing increasing attention for anti-TB vaccine
development. Here, we investigated the phenotypic and functional profiles of two novel
mycobacterial antigens Rv2251 and Rv2721c during T cell recall response via multi-color flow
cytometry.
Infective endocarditis is a life-threatening disease caused by bacterial infection of the endothelium and cardiac valves, either native or prosthetic. In the present work the role of the new microbiological techniques (techniques of detection and amplification of the subunit 16 ribosomal sRNA by means of the chain reaction of the polymerase in blood or tissue, fluorescent in situ hybridization, and matrix-assisted laser is reviewed desorption/ ionization time-of-flight mass spectrometry (MALDI-TOF MS) in the diagnosis of infective endocarditis.
Novel research aimed at finding a cure for AIDS requires animal models responding to human antiretroviral drugs. However, there have been few antiretrovirals cross-active against the simian viruses. In this study, we expanded the arsenal of drugs active against the simian retrovirus SIVmac251 and showed that this virus is inhibited by the protease inhibitor, darunavir, and the CCR5 blocker, maraviroc. Administration of these two drugs in combination with the reverse transcriptase inhibitors, tenofovir and emtricitabine, and the integrase inhibitor, raltegravir, resulted in prolonged plasma viral loads below assay detection limits, and, surprisingly, restricted the viral reservoir, a marker of which is viral DNA. We then decided to employ this multidrug regimen (termed “highly intensified ART”) in order to increase the potency of a previous strategy based on the gold drug auranofin, which recently proved able to restrict the viral reservoir in vivo. A short course of highly intensified ART following the previous treatment resulted, upon therapy suspension, in a remarkably spontaneous control of the infection, that may pave the way to a persistent suppression of viremia in the absence of ART. These results corroborate the robustness of the macaque AIDS model as a vanguard for potentially future treatments for HIV in humans.
Infective endocarditis is a life-threatening disease caused by bacterial infection of the endothelium and cardiac valves, either native or prosthetic. In the present work the role of the new microbiological techniques (techniques of detection and amplification of the subunit 16 ribosomal sRNA by means of the chain reaction of the polymerase in blood or tissue, fluorescent in situ hybridization, and matrix-assisted laser is reviewed desorption/ ionization time-of-flight mass spectrometry (MALDI-TOF MS) in the diagnosis of infective endocarditis.
Novel research aimed at finding a cure for AIDS requires animal models responding to human antiretroviral drugs. However, there have been few antiretrovirals cross-active against the simian viruses. In this study, we expanded the arsenal of drugs active against the simian retrovirus SIVmac251 and showed that this virus is inhibited by the protease inhibitor, darunavir, and the CCR5 blocker, maraviroc. Administration of these two drugs in combination with the reverse transcriptase inhibitors, tenofovir and emtricitabine, and the integrase inhibitor, raltegravir, resulted in prolonged plasma viral loads below assay detection limits, and, surprisingly, restricted the viral reservoir, a marker of which is viral DNA. We then decided to employ this multidrug regimen (termed “highly intensified ART”) in order to increase the potency of a previous strategy based on the gold drug auranofin, which recently proved able to restrict the viral reservoir in vivo. A short course of highly intensified ART following the previous treatment resulted, upon therapy suspension, in a remarkably spontaneous control of the infection, that may pave the way to a persistent suppression of viremia in the absence of ART. These results corroborate the robustness of the macaque AIDS model as a vanguard for potentially future treatments for HIV in humans.
Updated poster following beta v3 release. In preparation for Pharmacology Futures, Edinburgh Immunology Symposium and Word Congress of Pharmacology (Kyoto)
A Wellcome Trust-funded project to extend the Guide to PHARMACOLOGY (www.guidetopharmacology.org) to include data on key immunological data types and associate these to drugs and drug targets. Presented at the ELIXIR-UK All-Hand Meeting, Edinburgh, Nov 2017.
This ppt file represents a simple overview on what is antibody validation & how to validate an antibody before performing any research.
Used references are also included.
IUPHAR Guide to IMMUNOPHARMACOLOGY poster. Presented at the BSI Congress 2017, Brighton, UK (6th December 2017) and at Pharmacology 2017, London, UK (13th December 2017.
Current and Novel Immuno-Oncology Drug Evaluation Methods via Humanized Mouse...InsideScientific
Dr. Bin Xie discusses the current immuno-oncology drug development landscape, different humanized mouse models available for drug testing, and the investigation of potential mechanisms via imaging mass cytometry.
Since the first immune checkpoint blocker ipilimumab was approved by the US FDA in 2011, more drug companies have sought to develop their own immune therapy drugs. Humanized peripheral blood mononuclear cell (PBMC) reconstitution in immune deficient mice is becoming a valuable model for evaluating therapeutic antibodies, especially bispecific antibodies (BsAbs), which can mediate immune cells as well as target a tumor antigen.
However, this model has several drawbacks, including a limited dosing window due to graft-versus-host-disease and insufficient natural immune cell infiltration. This has hindered wide application of the model in the development of multiple immune checkpoint inhibitors or immune agonists.
To overcome these issues, LIDE has developed a unique human PBMC/cancer cell co-transfer model which can generate three-dimensional huPBMC-infiltrated tumor tissue for immunotherapy. This model has successfully been used to evaluate the biological function of several signaling proteins and biomarkers in multiple cancers, such as melanoma, breast cancer, and lung cancer.
In this webinar, Dr. Bin Xie discusses the current immuno-oncology drug development landscape, different humanized models available for drug testing, evaluates real-world case studies, and describes the investigation of potential mechanisms by imaging mass cytometry.
Key Topics Include:
- Introduction to immuno-oncology drug development and the importance of using humanized mouse models to address scientific questions
- Evaluation of current IO platforms and new methods from LIDE, including analysis of several case studies
- Understanding the spatiotemporal interaction between tissue-infiltrating immune cells and cancer cells via imaging mass cytometry
Poster on GtoImmuPdb presented at European Congress of Immunology (Amsterdam, Sep 2018). Overview of the main data types and features included in this extension to the IUPHAR/BPS Guide to PHARMACOLOGY
3. linking the isolated specificities to the Ag of interest. In this study,
we provide the proof of principle of such methodology. We focus
on the characterization of a selected high-affinity Ab directed
against the class B scavenger receptor CD36, expressed exclu-
sively on the CD8␣ϩ
subset of blood-derived conventional DCs.
Our results show that a fusion protein that contains the variable
regions of the Ab against CD36 linked to the model Ag OVA is
internalized and delivered to a processing pathway able to generate
both MHC class II and class I peptide complexes and to induce a
long-term protective response in vivo in the absence of any added
DC maturation stimuli.
Materials and Methods
Mice
Female C57BL/6 (B6) mice (6–7 wk old) were purchased from Harlan
Breeders. OVA-specific, TCR-transgenic OT-I and OT-II mice were pur-
chased from The Jackson Laboratory. CD45.1-congenic C57BL/6 (a gift
from P. Guermonprez, Institut Curie Paris, Paris, France) were bred to OT-I
mice to obtain OT-I/CD45.1. Animal care and treatment were conducted in
conformity with institutional guidelines in compliance with national and in-
ternational laws and policies (European Economic Community Council Di-
rective 86/609; OJL 358; December 12, 1987) and housed at the International
Centre for Genetic Engineering and Biotechnology animal house.
Abs and reagents
All mAb, if not specified otherwise, were purchased from BD Biosciences.
Anti-mouse CD40 (clone HM40-3) was obtained from eBioscience. Rabbit
polyclonal anti-CD36 Ab was purchased from Abcam. All reagents, if not
specified otherwise, were obtained from Sigma-Aldrich.
Cells
DCs were isolated from single-cell suspensions of lymph nodes or spleen
treated with 400 U/ml collagenase D (Roche) for 30 min followed by
enrichment (depletion of B, T, NK) or positive selection by CD11cϩ
beads
(Miltenyi Biotec). OT-II and OT-I cells were isolated from total lymph
node (LN) suspensions by negative selection using a MACS isolation kit
(Miltenyi Biotec). Langerhans cells and dermal DCs were isolated from the
epidermis as described previously (18).
Bone marrow-derived DCs (BMDCs) were generated using Fms-like
tyrosine kinase 3 ligand as described previously (18). DCs were used at day
10 and purity of CD11cϩ
was higher than 90%.
Phage display screening
For the screening, we used a highly diverse library (Ͼ1010
independent
clones) of single-chain Ab fragments (scFv) derived from samples of hu-
man PBLs (19). Phages were prepared following standard protocols. Phage
selection was performed on DCs isolated from spleen of C57BL/6 mice.
Briefly, 1013
CFU of phage were blocked in PBS/5% milk and allowed to
bind to 6 ϫ 106
DCs for 2 h at 4°C. After PBS washings, cells were
resuspended in IMDM and incubated at 37°C for 30 min. Extracellular
phages were inactivated with subtilisin (3 mg/ml) in buffer B (HBSS), 20
mM Tris, and 2 mM EDTA, pH 8) for 30 min. Cells were washed and lysed
in 1 ml of triethylamine (100 mM) for 8 min at room temperature (RT). The
lysate was neutralized with 0.5 ml of 1 M Tris-HCl (pH 7.4). Internalized
phages were recovered by infecting Escherichia coli (TG1) cells. Samples
were processed for quantitation of phage titer (19).
FACS and immunofluorescence analysis using phage
Phages (1012
CFU) were blocked in PBS/5% milk and allowed to bind to
BMDCs for 1 h at 4°C. For FACS analysis, phage binding was detected by
anti-M13 IgG mAb (Amersham Pharmacia) and FITC-conjugated anti-
mouse IgG (Kirkegaard &Perry Laboratories). For confocal analysis, cells
were plated on poly-L-lysine-treated coverslips and incubated either at 4 or
37°C for 30 min. After PBS washings, cells were fixed in 4% paraformal-
dehyde (PFA) and permeabilized with PBS/0.1% Triton X-100. The cov-
erslips were saturated with PBS/1% BSA (30 min at RT). Phage particles
were detected with rabbit anti-fD Bacteriophage Ab (Sigma-Aldrich) and
FITC-conjugated swine anti-rabbit IgG (DakoCytomation). Samples were
analyzed by confocal microscopy (Axiovert; Zeiss).
Immunoprecipitation/Western blotting
In brief, 107
BMDCs or BO9 T cells were surface biotinylated (EZ-Link
Sulfo-NHS-biotinylation kit) and lysed. Cell debris were spun out (12,000
rpm, 15 min) and supernatants were precleared with 10 g of anti-SV5tag
mAb (20) and 200 l of 50% protein A-Sepharose CL-4B beads (Amer-
sham Biosciences) for 2 h at 4°C. Biotinylated proteins were immunopre-
cipitated with scFv-2E5 or scFv-control (ctrl; 20 g/ml) for 2 h at 4°C,
followed by the addition of 5 g of anti-SV5tag mAb and 50 l of 50%
protein A-Sepharose CL-4B. Beads were washed three times in ice-cold
lysis buffer before SDS-PAGE analysis. Upon transfer on polyvinylidene
difluoride membranes (Millipore), the presence of biotinylated protein was
revealed by HRP-conjugated streptavidin (Amersham Biosciences).
In-gel digestion and mass spectrometry
In-gel digestion was performed as described elsewhere (21). Briefly, pro-
tein gel pieces were excised and tryptically digested with porcine trypsin.
After incubation overnight, gel pieces were centrifuged at 14,000 rpm for
5 min, then extracted once with 5% formic acid/50% acetonitrile and dried
by vacuum centrifugation. Mass spectrometry data were acquired working
in reflectron mode with a 4800 MALDI TOF/TOF. A 0.5-l aliquot of the
peptide solution was mixed with 0.5 l of a-cyano-4-hydroxycinnamic acid
matrix and subjected to MALDI analysis. Mass spectrometry data were
subjected to database searching using Mascot (Matrix Science) against
SwissProt database. Up to one missed tryptic cleavage and optional me-
thionine oxidation and carbamidomethylation was considered. Mass accu-
racy was limited to 80 ppm.
Production of scFv-OVA proteins
The scFv isolated from the library and an irrelevant scFv (scFv-ctrl) were
engineered into a small immunoprotein (SIP) format by cloning them be-
tween the secretory signal sequence from a mouse Ig H chain and the third
constant domain of human IgG1 (␥1-CH3) (22). The sequence encoding
for full-length OVA was cloned in-frame downstream of the ␥1-CH3 do-
main, followed by a His6 tag. For comparison, the DNA fragment encoding
for V regions of anti-DEC-205/CD205 rat mAb (clone NLDC-145) were
amplified and cloned into a similar SIP-OVA format. SIP-OVA-H6 pro-
teins were transiently expressed in 293T cells after calcium-phosphate
transfection. Proteins were purified by affinity chromatography using a
poly-H6 tag purification system (Ni-NTA His Bind Resin; Novagen) and
quantified by Coomassie staining and Western blot using a rabbit anti-
OVA Ab (Abcam). The recombinant scFv-OVA proteins were tested for
the presence of endotoxin by the chromogenic Limulus amebocyte lysate
assay (Charles River ENDOSAFE). The levels of endotoxin were below
0.05 endotoxin units/g in all of the scFv-OVA proteins used in vivo or in
culture.
ScFvCD36
-OVA internalization assay
DCs were incubated at 4°C for 1 h with scFvCD36
-OVA or scFvctrl
-OVA,
washed and then either kept at 4°C or incubated at 37°C for 15, 30, or 60
min. For FACS analysis, the level of scFvCD36
-OVA remaining at the cell
surface was detected using rabbit anti-OVA and FITC-conjugated goat
anti-rabbit IgG (Kirkegaard & Perry Laboratories). The median fluorescent
values of OVAϩ
cells was determined and used to calculate the percent
remaining scFvCD36
-OVA at the surface, with 1 h at 4°C incubated cells
taken as 100%. For confocal analysis, after binding at 4°C for 1 h, cells
were washed, plated on poly-L-lysine-treated coverslips, and incubated ei-
ther at 4 or 37°C for 30 min. Membranes were counterstained with 5 g/ml
cholera toxin subunit B-FITC, washed, fixed in 4% PFA, and permeabil-
ized with 0.02% saponin. ScFvCD36-OVA was detected with Alexa Fluor
594-F(abЈ)2 goat anti-human IgG (Molecular Probes).
In vitro T cell proliferation assay
DCs were pulsed with graded doses of scFv-OVA proteins or soluble OVA
(Worthington Biochemical) for 3 h at 37°C. After washing, 1 ϫ 105
OT-I
or OT-II cells were cocultured with Ag-pulsed DCs in round-bottom 96-
well plates (1 DC:5 T cell ratio). After 48 h, [3
H]thymidine (1Ci; Am-
ersham Biosciences) was added for 18 h, and incorporation was measured
by liquid scintillation counting after collection of cells on a glass fiber filter
with and automatic cell harvester (Tomtec).
Adoptive transfer and T cell proliferation responses
OT-I or OT-II cells were labeled with 7 M CFSE according to the man-
ufacturer’s instructions. C57BL/6 mice were i.v. injected with 1–2 ϫ 106
OT-I or OT-II cells followed by injection in the footpad of scFv-OVA
proteins. Three days later, lymph node cell suspensions were stained for
CD8 or CD4, respectively, and the CFSE dilution was evaluated by FACS.
To evaluate long-term OT-I persistence, 1.5 ϫ 106
OT-I/CD45.1 were
injected i.v. into recipient CD45.2 hosts immunized with scFv-OVA pro-
teins. Twelve days later, spleen cell suspensions was stained for surface
3202 TARGETING CD36 INDUCES CROSS-PRESENTATION BY DCs
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4. CD45.1 and CD8. Intracellular IFN-␥ production was evaluated using
splenocytes or blood cells cultured for 5 h with 1 M OVA257–264 peptide
in the presence of brefeldin A (BD Biosciences). Cells were stained with
anti-mouse CD8 and CD3 Abs for 25 min at 4°C. After fixation with 2%
PFA, cells were stained for intracellular IFN-␥ in PermWash solution (BD
Biosciences) for 30 min at 4°C.
In vivo cytotoxicity assay
Naive syngenic splenocytes were pulsed with OVA257–264 peptide (5 M)
for 1 h at 37°C, washed, and labeled with 5 M CFSE. Nonpulsed
control splenocytes were labeled with low concentration of CFSE (0.5
M). CFSEhigh
and CFSElow
cells were mixed at 1:1 ratio (7 ϫ 106
cells
each) and injected i.v. into mice. After 15 h, the numbers of CFSEϩ
cells
in spleen and lymph nodes were determined by FACS.
Anti-OVA Ab ELISA
Ninety-six-well Maxisorp ELISA plates (Nunc) were coated overnight
with 3 g/ml OVA. Plates were washed and blocked in PBS/1% BSA/
0.1% Tween 20. Sera collected from the clotted blood of immunized mice
were serially diluted in blocking solution and incubated for 1 h at RT.
Plates were washed and Ab binding was detected using HRP-conjugated
anti-mouse IgG Ab (Kirkegaard & Perry Laboratories) followed by tetra-
methylbenzidine peroxidase substrate (Sigma-Aldrich).
Tumor rejection experiments
Mice were injected s.c. on days 0 and 7 with 0.5 g of scFvCD36
-OVA or
scFvCtrl
-OVA. Seven or 30 days after the second immunization, each
mouse was challenged s.c. with 2 ϫ 105
EG7-OVA cells (ATCC CRL-
2113). Tumor size was measured with a caliper ruler at different time
points after the challenge. Average size is expressed in cubic centimeters
using the formula V ϭ (length ϫ width2
)/2. Significance of protection was
evaluated using a two-tailed Student t test.
Results
Selection of internalizing phages by panning on DCs
We used a phage display library of single-chain Ab fragments
(scFv) to isolate Abs specific for surface molecules of mouse DCs.
We set up a whole cell panning procedure to specifically select
Abs able to trigger the internalization of the bound DC receptors.
Multiple rounds of positive selection using a highly diverse scFv-
phage library (Ͼ1010
independent clones) (19) were performed on
CD11cϩ
cells purified from the spleen of C57BL/6 mice. To se-
lectively recover and propagate only the internalized phages, we
used subtilisin, a protease that inactivates extracellular phages by
cleavage of the phage protein pIII, thus rendering the phage par-
ticles noninfectious (23) (Fig. 1A). To evaluate enrichment for spe-
cific binders, we analyzed by flow cytometry the pool of clones
recovered after each round of selection. The polyclonal scFv-
phage population from the third round of panning, compared with
the pools of the first and second round, showed significantly in-
creased binding on DCs (Fig. 1B) but not on an irrelevant cell type
(A20) used as control (data not shown). We determined by immu-
nofluorescence that the polyclonal scFv-phage population from the
third round of panning was internalized by DCs upon incubation at
37°C, suggesting that most of the isolated ligands target endocytic
receptors (Fig. 1C). Randomly picked individual phage clones
from the third round of panning were analyzed by DNA finger-
printing and DNA sequencing. Sixty percent of the 40 clones an-
alyzed showed the same fingerprinting pattern (data not shown).
DNA sequencing confirmed the presence of a dominant clone, des-
ignated scFv-2E5, which was further analyzed.
Characterization of the selected scFv
The spleen-derived DC populations used for the phage selection
procedure comprises three different subtypes: CD4Ϫ
CD8ϩ
,
CD4ϩ
CD8Ϫ
, and double-negative (CD4Ϫ
CD8Ϫ
) DCs. Therefore,
we asked whether the Ag recognized by scFv-2E5 was expressed
on a specific DC subset. We evaluated by flow cytometry the bind-
ing of scFv-2E5 on splenic DCs. When we analyzed CD11chigh
DCs, we found a tight correlation between CD8␣ expression and
binding of scFv-2E5, resulting in two predominant populations of
conventional DCs: CD8␣ϩ
scFv-2E5ϩ
and CD8␣Ϫ
scFv-2E5Ϫ
.
The same type of correlation was evident on DCs isolated from
LNs (data not shown). Further analysis on plasmacytoid and tis-
sue-derived DCs (Langerhans cells and dermal DCs) did not show
any specific staining (Fig. 2). We therefore concluded that the Ag
recognized by scFv-2E5 is uniquely expressed by the CD8␣ϩ
sub-
set of conventional DCs, the main subtype involved in Ag
cross-priming.
When binding specificity was investigated for other hemopoietic
cell types present in the spleen, only macrophages and a percent-
age (30%) of B cells were positive, whereas both monocytes and
T lymphocytes were negative (Fig. 2). These data indicate that
scFv-2E5 recognizes a cell surface marker specifically expressed
by APCs.
To characterize the molecule targeted by scFv-2E5, we con-
ducted immunoprecipitation experiments using extracts of surface
biotinylated BMDCs or T cells (BO9 T lymphocytes) as a negative
control. These experiments identified a DC-specific protein with an
apparent molecular mass of 90 kDa (Fig. 3A). By MALDI mass
spectrometry, we identified the immunoprecipitated band as the
mouse class B scavenger receptor CD36. The band immunopre-
cipitated by scFv-2E5 reacted in Western blot with a commercially
available anti-CD36 Ab (Fig. 3B). Moreover, the full-length cDNA
sequence of CD36 was amplified and transfected in 293T cells.
FIGURE 1. Selection of internalizing phages by panning on DCs. A,
Schematic representation of the panning procedure. Phages were allowed
to bind to DCs and to be internalized by incubation at 37°C. Noninternal-
ized phages were inactivated by subtilisin treatment. DCs were lysed to
recover internalized phages by infection of E. coli cells. Rescued phage-
mids were used for the next round of selection. B, Flow cytometry analysis
on DCs to monitor the enrichment of binders in the polyclonal phage prep-
arations from each round of selection. C, Internalization of phage particles
by DCs incubated with the original library or the pool of phages from the
third round of panning (Sel III) using the anti-pIII Ab and a FITC-conju-
gated secondary Ab.
3203The Journal of Immunology
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5. Flow cytometry analysis showed that scFv-2E5 specifically stained
CD36-transfected but not mock-transfected 293T cells (Fig. 3C).
These results demonstrate that scFv-2E5 recognizes an epitope in
the extracellular portion of CD36.
Targeting Ags to CD36 induces presentation of MHC class I
and MHC class II epitopes in vitro
CD36 belongs to the class B scavenger receptors family. It is a
multi-ligand receptor involved in cellular adhesion and lipid me-
tabolism. In vitro studies with human DCs have suggested a pos-
sible role of CD36 in cross-presentation of apoptotic material (24).
However, this molecule is not essential for either cross-presenta-
tion or cross-tolerance of cell-associated Ags in mice (25, 26).
Nevertheless, a role for CD36 in other pathways of cross-presen-
tation cannot be excluded.
To investigate whether CD36 could be used as a targeting mol-
ecule for the induction of T cell immune response by DCs, the
scFv-2E5 was engineered into the SIP format, a molecule that
contains, downstream of the scFv, the third constant domain of
human IgG1 (␥1-CH3), thus producing a dimeric scFv-2E5 (22).
The C terminus of the SIP-2E5 was fused in-frame to the full-
length sequence of OVA, followed by a His6 tag, to generate the
dimeric recombinant protein that we named scFvCD36
-OVA (Fig.
4A). As a control, we constructed a similar recombinant containing
an irrelevant scFv (scFvctrl
-OVA). Both proteins were produced by
transient transfection in 293T cells (ϳ1–2 mg/L) and purified from
culture supernatants by anti-His6 tag affinity chromatography.
Analysis by Western blot of the purified proteins revealed, for each
of them, a single band of the expected molecular mass (90 kDa;
Fig. 4B).
To study the endocytosis of scFvCD36
-OVA, we performed a
flow cytometry-based internalization assay. scFvCD36
-OVA was
rapidly internalized with ϳ70% of the protein removed from the
cell surface within 30 min at 37°C (Fig. 4C). These data were
confirmed by immunofluorescence: scFvCD36
-OVA was efficiently
internalized at 37°C while it remained associated to the plasma
membrane upon incubation at 4°C (Fig. 4D).
To investigate the capacity of scFvCD36
-OVA to deliver OVA-
derived peptides to the MHC class I and MHC class II pathways of
Ag presentation, we performed in vitro proliferation assays using
OT-I and OT-II cells. CD11cϩ
DCs isolated from the spleen of
FIGURE 3. ScFv-2E5 recognizes an epitope in the extracellular portion
of the scavenger receptor CD36. Western blot of extracts from cell surface-
biotinylated DCs or BO9 T cells, immunoprecipitated with scFv-2E5 (2E5)
or an irrelevant scFv (ctrl) and revealed with streptavidin-HRP (A) or with
an anti-CD36 Ab (B) as indicated. C, Binding of scFv-2E5 or scFv-ctrl to
293T cells transiently transfected with the cDNA-encoding mouse CD36.
FIGURE 4. Internalization of the recombinant anti-CD36-Ag fusion. A,
Schematic representation of the scFv-OVA recombinant molecule. B, Western
blot (anti-OVA) of recombinant Ag molecules: soluble OVA (lane 1), scFvCD36
-
OVA (lane 2), and scFvctrl
-OVA (lane 3). C, Time course of scFvCD36
-OVA
internalization. After binding at 4°C, DCs were incubated either at 37 or at 4°C for
the indicated time points. The plot shows the ratio of remaining surface-bound
scFvCD36
-OVA at the two temperatures, measured with an anti-OVA Ab. Data
show means of duplicate measurements. D, Confocal microscopy of scFvCD36
-
OVA internalization by DCs. After binding at 4°C, cells were washed and
incubated for 30 min at 4 or 37°C as indicated. Fixed and permeabilized DCs
were stained with Alexa 594-conjugated anti-human Ab and FITC-conjugated
cholera toxin subunit B to counterstain plasma membranes. Images from the
central Z section of representative cells are shown.
FIGURE 2. Binding profile of scFv-2E5 on splenic DCs and hemopoi-
etic cells. DCs from the spleen of C57BL/6 mice were labeled with scFv-
2E5 and a panel of different markers gating on: CD11chigh
and CD8␣ϩ/Ϫ
for conventional DCs and CD11cint
and B220ϩ
for plasmacytoid DCs.
Langerhans cells and dermal DCs were isolated from the epidermis and
identified by gating on CD11cϩ
cells. Monocytes were defined by gating
on Ly-6Chigh
, CD11bhigh
, and CD11cϪ
cells, macrophages as F4/80ϩ
and
Ly-6Cint
, B lymphocytes by expression of B220 and T cell by expression
of CD3. Filled line, scFv-2E5.
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6. C57BL/6 mice were incubated with graded concentrations of the
recombinant scFvCD36
-OVA or scFvctrl
-OVA proteins or soluble
OVA and then cocultured with OT-I and OT-II cells. T cell pro-
liferation was evaluated 3 days later by [3
H]thymidine incorpora-
tion. scFvCD36
-OVA induced OT-I and OT-II proliferation at a
concentration as low as 20 ng/ml, whereas no proliferation was
detected even at the highest concentration (1600 ng/ml) of scFvctrl
-
OVA. Relative to soluble OVA, uptake via CD36 increased the
efficiency of presentation by at least 400-fold for the MHC class I
epitope (Fig. 5A) and by 300-fold for the MHC class II epitope
(Fig. 5B). These results demonstrate that targeting CD36 on
splenic DCs increases the efficiency of presentation of protein Ags
on MHC class II and, more importantly, is able to deliver OVA to
the MHC class I pathway of Ag cross-presentation.
CD36 mediates CD4ϩ
and CD8ϩ
naive T cells activation in
vivo
A major drawback of the current DC-based clinical approaches
derives from the necessity of their ex vivo manipulation. Few stud-
ies have addressed the possibility to use Abs to specifically target
and activate DCs in vivo (4, 11, 12). We asked whether targeting
OVA Ag to CD11cϩ
CD8aϩ
DCs via CD36 would be convenient
to induce potent immune responses in vivo.
To investigate the ability of scFvCD36
-OVA to induce prolifer-
ation of OVA-specific CD4ϩ
T cells, mice were adoptively trans-
ferred with CFSE-labeled OT-II cells followed by immunization
with graded doses of scFvCD36
-OVA or scFvctrl
-OVA. T cell pro-
liferation was evaluated in the draining LNs at day 3 upon injection.
We detected OT-II proliferation only in LNs of mice immunized with
scFvCD36
-OVA (200 ng), whereas priming with a higher amount of
scFvctrl
-OVA (500 ng) did not elicit OT-II activation (Fig. 6A). These
data indicate that targeting of CD36 enhances the efficiency of MHC
class II peptide Ag presentation in vivo.
We next focused on the ability to trigger CTLs since this is
particularly challenging and would be valuable for the design of
effective vaccines. To evaluate the relative ability of DCs to cross-
present OVA in vivo, we performed a comparative study with a
well-characterized system of Ag delivery to DCs, the DEC-205
receptor (4, 5). The V regions of the anti-DEC-205 mAb (NLDC-
145) were amplified and engineered in the same format as
scFvCD36
-OVA. The recombinant scFvDEC205
-OVA protein main-
tained the same binding specificity as the original anti-DEC mAb
(data not shown). Mice were adoptively transferred with CFSE-
labeled OT-I cells and s.c. injected with increasing amounts of
scFvCD36
-OVA, scFvDEC205
-OVA, or scFvctrl
-OVA. Ag-specific
T cell proliferation was determined in the draining LNs at day 3
upon transfer. Practically all of the OT-I cells in LNs of scFvCD36
-
OVA-immunized mice entered cell cycle and underwent up to six
divisions after a dose of just 100 ng of OVA protein, whereas
immunization with the highest dose of scFvctrl
-OVA (300 ng) did
not induce naive OT-I cells to divide. Immunization with
scFvDEC205
-OVA, however, was more efficient in inducing entry
in the cycle since as little as 10 ng of protein induced extensive T
cell proliferation (Fig. 6B). Altogether these data indicate that de-
livery of Ags through CD36 in vivo promotes uptake and process-
ing for peptides presentation on MHC class II and class I
molecules.
To evaluate a possible contribution of other APCs to presenta-
tion of OVA upon immunization with scFvCD36
-OVA, we com-
pared cross-presentation mediated by DCs and B cells that express
high levels of CD36. DCs from spleen and LNs and CD19ϩ
B cells
were pulsed in vitro with different amounts of scFvCD36
-OVA,
FIGURE 5. In vitro targeting of CD36 on splenic DCs. Proliferation
([3
H]thymidine uptake) of OT-I cells (A) or OT-II cells (B) cocultured for
72 h with spleen-derived DCs pulsed with different concentrations of
scFvCD36
-OVA (F), scFvctrl
-OVA (ࡗ), or OVA alone (Œ).
FIGURE 6. CD36-targeted Ag accesses the exogenous and cross-pre-
sentation pathways in vivo. C57BL/6 mice were injected i.v. with 1 ϫ 106
CFSE-labeled OT-II (A) or OT-I (B) T cells and subsequently injected s.c.
with graded doses of scFvCD36
-OVA (CD36), scFvctrl
-OVA (ctrl), or
scFvDEC205
-OVA (DEC205) as indicated. T cell proliferation was mea-
sured at day 3 in draining LNs. Histograms represent the CFSE dilution
profile of OT-II cells (A) and OT-I cells (B) at day 3.
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7. scFvDEC205
-OVA, or scFvctrl
-OVA and cocultured with CFSE-la-
beled OT-I cells. At day 3, we evaluated T cell proliferation as
well as IL-2 production. Results show that only DCs are able to
efficiently take up and present OVA to OT-I cells. B cells did not
stimulate T cell proliferation above the background levels, even at
Ag and cell doses 10-fold higher than those required to observe
proliferation by DCs. scFvctrl
-OVA did not cause T cell activation
even at the highest Ag dose (1 g), whereas scFvDEC205
-OVA was
at least twice as efficient as CD36 in inducing T cell proliferation
(Fig. 7). Only T cells primed by DCs secreted IL-2 (data not
shown). These results indicate that targeting the CD36 receptor
induces efficient cross-presentation exclusively in DCs.
ScFvCD36
-OVA immunization induces differentiation of naive
OT-I cells into effector CTLs in the absence of adjuvancy
Procedures for targeting Ags to DCs in vivo should, in addition,
induce the activation of DCs, since targeting Ags to immature DCs
may lead to tolerance (4, 5).
To evaluate the long-term effects of targeting Ags to the CD36
receptor on DCs, we followed the fate of the OT-I cells that pro-
liferated upon immunization with scFvCD36
-OVA and scFvDEC205
-
OVA, for comparison. Naive C57BL/6 mice were adoptively
transferred with 1.5 ϫ 106
OT-I cells and s.c. injected with 300 ng
of scFvCD36
-OVA, scFv scFvDEC205
-OVA, or scFvctrl
-OVA either
in the presence or absence of adjuvant (anti-CD40 mAb). At day
12 upon transfer, we evaluated the expansion of Ag-specific ef-
fector T cells in the spleen. In mice immunized with scFvCD36
-
OVA without adjuvant, we found a 3- to 4-fold expansion of OT-I
cells, relative to mice injected with scFvctrl
-OVA, or PBS (Fig. 8A)
and a high proportion of cells secreted IFN-␥ upon in vitro re-
stimulation, indicating acquisition of effector functions (Fig. 8B).
Coadministration of anti-CD40 mAb with scFvCD36
-OVA did not
significantly increase T cell expansion nor the proportion of IFN-
␥-secreting cells. In contrast, immunization with scFvDEC205
-
OVA, despite initial T cell proliferation, did not induce detectable
expansion of Ag-specific effector cells unless codelivered with the
anti-CD40 mAb. The few OT-I cells that persisted were unable to
secrete IFN-␥, but were rescued by coadministration of adjuvant
FIGURE 7. Cross-presentation by DCs and B cells. Proliferation of
CFSE-labeled OT-I T cells, cocultured for 3 days with 5 ϫ 104
DCs (iso-
lated from spleen or LNs) or B cells (5 ϫ 104
or 5 ϫ 105
), preincubated
with scFvCD36
-OVA (CD36), scFvctrl
-OVA (ctrl), or scFvDEC205
-OVA
(DEC-205). Plots represent the percentage of OT-I cells that underwent
proliferation.
FIGURE 8. CD36-targeting to steady-state DCs induces long-lasting CTLs. In brief, 1.5 ϫ 106
OT-I (CD45.1ϩ
) T cells were adoptively transferred into
CD45.2ϩ
C57BL/6 mice followed by immunization with 0.3 g of scFvCD36
-OVA (CD36), scFvDEC205
-OVA (DEC-205), or scFvctrl
-OVA (ctrl), with or
without the addition of anti-CD40 Ab (25 g). Spleens were harvested at day 12 by flow cytometry: A, the percentage of persistent CD45.1ϩ
CD8ϩ
cells
(OT-I) and B, their ability to produce IFN-␥ following restimulation in vitro. C, In vivo cytotoxicity assay. Mice were treated as above. The ability of primed
OT-I cells to kill an Ag-specific target cell was evaluated at day 12 by injecting mice with a mix of CFSE-labeled syngeneic splenocytes pulsed (CFSEhigh
)
or not pulsed (CFSElow
) with the OVA257–264 class I peptide. Fifteen hours postinjection, the ratio between CFSEhigh
and CFSElow
(r) was evaluated as
a measure of specific CTL activity.
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8. (Fig. 8, A and B). Thus, according to a previous report (4), Ag
targeting to CD8ϩ
DCs via the DEC205 receptor requires a mat-
uration stimulus to induce immunity. Instead, targeting the same
DC subsets via CD36 induces T cell expansion per se. It should be
noticed that T cell expansion was higher in mice primed with
scFvCD36
-OVA than in mice immunized with scFvDEC205
-OVA
plus adjuvant.
To directly evaluate the effector functions of OT-I cells gener-
ated upon targeting Ags to CD36, we performed an in vivo cyto-
toxicity assay. Mice were adoptively transferred with 1.5 ϫ 106
OT-I cells and then s.c. injected with 200 ng of scFvCD36
-OVA or
scFvctrl
-OVA either with or without anti-CD40 Ab. At day 12 after
immunization, we injected a mixture of OVA peptide-pulsed and
nonpulsed syngeneic splenocytes to assess the cytolytic activity of
in vivo-primed OT-I cells. Effective cytotoxicity was observed
only in LNs and spleen of mice immunized with scFvCD36
-OVA
regardless of the coadministration of anti-CD40. In mice primed
with scFvCD36
-OVA, ϳ90% of the peptide-pulsed targets were
eradicated from LNs, while a small proportion of target cells could
still be detected in the spleen (Fig. 8C). Together, these data dem-
onstrate that a single immunization with scFvCD36
-OVA is suffi-
cient to induce a durable formation of effector T cells even in the
absence of any additional maturation stimulus.
Targeting a tumor Ag to CD36 elicits a protective immune
response
Having demonstrated the ability of scFvCD36
-OVA to induce prim-
ing and differentiation of adoptively transferred OT-I cells into
effector T cells, we next sought to determine whether targeting
OVA to CD36 could activate, as well, an endogenous Ag-specific
T cell response.
To this purpose, naive C57BL/6 mice were immunized twice in
14 days with 500 ng of scFvCD36
-OVA or scFvctrl
-OVA. Seven
days after the second immunization, lymphocytes isolated from
blood were tested for the ability to produce IFN-␥ upon in vitro
restimulation with OVA257–264 peptide. As shown in Fig. 9A, mice
injected with scFvCD36
-OVA, but not with scFvctrl
-OVA, devel-
oped an Ag-specific CTL response as demonstrated by the per-
centage (4%) of IFN-␥-secreting CD8ϩ
T cells. In parallel, we
examined the humoral immune response by measuring the titers of
OVA-specific IgG Abs by ELISA. High titers of anti-OVA-
specific Abs were found in mice primed with scFvCD36
-OVA, but
not with scFvctrl
-OVA (Fig. 9B).
We next determined whether the activation of the endogenous
OVA-specific T cell response confers protection against the graft
of EG7-OVA tumor cells. Mice were immunized at days 0 and 7
FIGURE 9. Activation of endogenous T cells by tar-
geting CD36. A, Induction of OVA-specific CD8ϩ
T
cells. Mice were immunized twice at 1-wk intervals
with scFvCD36
-OVA, scFvctrl
-OVA (0.5 g), or left un-
treated. Seven days later, lymphocytes isolated from
blood were restimulated in vitro with the OVA257–264
peptide. Dot plots show one representative IFN- ␥ se-
cretion profile in the different groups (left panel). Data
expressed as means of IFN-␥-secreting CD8ϩ
T cells
(right panel). B, OVA-specific IgG Ab titers from sera
of animals immunized as in A. Seven days after the
second immunization, sera of mice (n ϭ 5) were col-
lected and anti-OVA IgG Ab titer was determined by
ELISA. Data are expressed as means of OVA-specific
IgG Ab from serially diluted sera of mice immunized
with scFvCD36
-OVA (F) or scFvctrl
-OVA (f).
FIGURE 10. CD36-targeted vaccine induces tumor
immunity. C57BL/6 mice were immunized s.c. on days
0 and 7 with 0.5 g of scFvCD36
-OVA (F), scFvctrl
-
OVA (f), or left untreated (). Seven days (A) or 30
days (B) after the second immunization, mice were chal-
lenged s.c. with 2 ϫ 105
EG7-OVA cells. Tumor
growth was monitored once a week with a caliper. Av-
erage tumor size of the six mice (A) or five mice (B)
group was expressed in cubic centimeters using the for-
mula V ϭ (length ϫ width2
)/2. Significance of protec-
tion was evaluated using a two-tailed Student t test.
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9. with 500 ng of scFvCD36-OVA or scFvctrl-OVA and challenged
1 wk after the last immunization, a time point that corresponds to
the peak of effector T cell expansion. To assess vaccine memory,
we performed a parallel experiment by challenging mice 1 mo
after the last boost. Results in Fig. 10 show that mice immunized
with scFvCD36-OVA exhibited a significant tumor growth inhi-
bition compared to mice vaccinated with scFvctrl-OVA, both
when the challenge was performed at early (7 days; pϭ0.0005) or
later (30 days) time points ( pϭ0.008). Altogether these results
demonstrate that targeting DCs via CD36 with low doses of Ag
and without adjuvant is capable of inducing endogenous T cell
responses that delay the growth of an Ag-specific tumor.
Discussion
The selection of an appropriate Ab is crucial to improve Ag tar-
geting to DCs for vaccination purposes. In this study, we report the
successful application of a procedure to select, from a filamentous
phage display library, scFv Ab fragments internalized by DCs. We
isolated a panel of scFvs able to bind to DC surface molecules and
capable of triggering internalization of the bound receptor. We
focused on one binder specific for the CD8␣ϩ
subset of conven-
tional DCs and whose target was characterized as the class B scav-
enger receptor CD36. To create an Ab-based vaccine, we rescued
the sequence of the Ab V regions isolated from the screening and
we fused them to an antigenic unit. Targeting Ag to DCs via CD36
greatly enhanced the efficiency of Ag presentation to T cells on
both MHC class I and class II products. In vivo, immunization
with the anti-CD36 recombinant vaccine induced long-term T cell
expansion and tumor protection.
These results unveil a new function for the CD36 receptor in
adaptive immunity. CD36 has been implicated in multiple biolog-
ical processes that define it as a multi-ligand scavenger receptor,
mainly involved in cellular adhesion and lipid metabolism (27).
Few reports have addressed the role of CD36 in APCs. At first,
CD36 was shown to be involved in phagocytosis of apoptotic bod-
ies (24). Subsequent studies indicated that CD36 function is re-
dundant since CD36Ϫ/Ϫ
APC were fully competent to carry out
cross-presentation of Ags derived form apoptotic cells in vivo (25,
26). Most recent data suggest that CD36 is selectively implicated
in the presentation of Ags derived from apoptotic bodies on MHC
class II molecules (28). In this study, we show that CD36-mediated
endocytosis of soluble Ags increases at least 300-fold the delivery
of peptides to the classical MHC class II and the cross-presentation
pathways (Fig. 5). This property is unique to DCs since B cells,
that express the receptor and bind the recombinant Ab, were un-
able to cross-present the OVA epitope to T cells (Fig. 7).
This new mechanism for Ag cross-presentation is particularly
relevant to understand the biology of blood-derived CD8␣ϩ
DCs.
Among lymphoid organ resident DCs, those expressing the CD8␣
marker are clearly the most efficient at cross-presenting cellular
(3), soluble (2, 10), and pathogen-associated Ags (29, 30). Current
data suggest that the ability of CD8␣ϩ
DCs to process exogenous
Ags for presentation on MHC class I molecules may be regulated
at two levels: 1) differential expression of receptors for endoph-
agocytosis and 2) expression of a specialized pathway to promote
the generation of MHC class I peptides (31). Ags captured via the
C-type lectin DEC205 and the mannose receptors that are both
CD8␣ϩ
specific are cross-presented (5, 12). We provide evidence
of another CD8␣ϩ
-specific receptor that induces cross-presenta-
tion of endocytosed Ags. Thus, it is tempting to speculate that
targeting any endocytic receptors on CD8␣ would lead to cross-
presentation, although with different efficiencies.
For vaccination purposes, the targeting molecule should be able
to generate both classes of MHC peptide complexes at the cell
surface and provide additional signals to achieve full activation of
naive T lymphocytes. Indeed, a clear comparison of the long-term
effects triggered by different receptors remains an open issue. The
C-type lectin DEC205 has been the prototype DC target receptor to
induce immune responses against various Ags (7, 32, 33). How-
ever, targeting DEC205 leads to tolerance unless a second matu-
ration signal such as CD40L is provided (5). We demonstrate that
when targeting CD36, such an additional signal is dispensable for
effector CD8ϩ
differentiation. It is interesting to note that when
comparing the proliferation of Ag-specific CD8ϩ
T cells induced
by targeting DEC205 or CD36 via identical recombinant mole-
cules, the early response was stronger in the case of DEC205. The
dichotomy arises only at day 12 when almost no expanded T cells
persisted in animals immunized with anti-DEC205, whereas effi-
cient expansion is obtained in the case of CD36. How can these
divergent outcomes be explained? We can exclude that long-term
accumulation of cytotoxic T cells by immunization through CD36
reflects higher levels of TCR engagement. Rather the T cell pro-
liferation profile at day 3 indicates that targeting DEC205 gener-
ates higher amounts of MHC class I peptide complexes at the cell
surface. A second possibility is that CD36 but not DEC205 pro-
vides per se a DC maturation signal. Although we did not inves-
tigate the direct effect of the scFv anti-CD36 on the maturation
state of DCs in vivo, several reports have shown that engagement
of CD36 can modulate DC maturation and functions (28, 34, 35).
It would be interesting to investigate whether engagement of CD36
in our model may activate proinflammatory responses by coengag-
ing TLRs at the cell surface, as shown to occur during the uptake
of Staphylococcus aureus (36).
We tested the potential of immunization via CD36 in a tumor
rejection model. Mice immunized with low doses (0.5 g) of anti-
CD36-OVA in the absence of adjuvancy mounted an endogenous
T cell response that was sufficient to protect them from the growth
of an Ag-specific tumor. Although further studies are needed to
fully evaluate the efficiency of CD36 immunization against other
tumor-associated Ags, these results indicate that targeting CD36
may be a valuable strategy to induce immunity without the need to
coadminister immunostimulatory molecules.
In summary, we have identified a novel approach to initiate
CD8ϩ
T cell-mediated immunity by targeting endogenous DCs in
vivo and a new tool to specifically study the properties of the
unique subset of conventional CD8␣ϩ
DCs.
Acknowledgments
We are grateful to A. Bradbury and D. Sblattero for the phage display
library, F. Odreman and M. Hampel for MALDI-TOF analysis, and
M. Sturnega for technical assistance.
Disclosures
The authors have no financial conflict of interest.
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