The authors have declared that no competing interests exist.
Conceived and designed the experiments: JM FE PH YG. Performed the experiments: JM FE RO AL YG. Analyzed the data: JM FE PH SN FJ YG. Contributed reagents/materials/analysis tools: PH ED PMK. Wrote the paper: JM FE PH PMK FJ YG.
Antitumor vaccination using synthetic long peptides (SLP) is an additional therapeutic strategy currently under development. It aims to activate tumor-specific CD8+ CTL by professional APCs such as DCs. DCs can activate T lymphocytes by MHC class I presentation of exogenous antigens - a process referred to as “cross-presentation”. Until recently, the intracellular mechanisms involved in cross-presentation of soluble antigens have been unclear. Here, we characterize the cross-presentation pathway of SLP Melan-A16–40 containing the HLA-A2-restricted epitope26–35 (A27L) in human DCs. Using confocal microscopy and specific inhibitors, we show that SLP16–40 is rapidly taken up by DC and follows a classical TAP- and proteasome-dependent cross-presentation pathway. Our data support a role for the ER-associated degradation machinery (ERAD)-related protein p97/VCP in the transport of SLP16–40 from early endosomes to the cytoplasm but formally exclude both sec61 and Derlin-1 as possible retro-translocation channels for cross-presentation. In addition, we show that generation of the Melan-A26–35 peptide from the SLP16–40 was absolutely not influenced by the proteasome subunit composition in DC. Altogether, our findings propose a model for cross-presentation of SLP which tends to enlarge the repertoire of potential candidates for retro-translocation of exogenous antigens to the cytosol.
The notion that the immune system can recognize and mount a response against tumors was initially postulated by Coley
The adaptive immune response depending on CTL involves TAA expression by tumor cells and requires TAA presentation by professional APCs. Among professional APC, DCs possess the unique ability, via co-stimulatory signals, to activate naive T lymphocytes in secondary lymphoid organs
Current immunotherapy strategies are designed to provide either passive or active immunity against malignancies by harnessing the immune system to target tumors
A new immunotherapy strategy has emerged based on synthetic long peptides (SLP). SLP are usually 25–50 amino acids long and contain antigenic epitopes that require endocytosis and processing by professional APC such as DCs
In this study, we designed a SLP from the Melan-A/MART-1 TAA. This SLP of 25 amino acids covers positions 16 to 40 of Melan-A/MART-1 (SLP16–40) and includes the A27L modification which allows a better anchoring of the immunodominant Melan-A/MART-1 26–35 epitope to the HLA-A*0201 molecule
Culture medium RPMI 1640 (Gibco BLR, Gaithersburg, MD) was supplemented with penicillin-streptomycin (100 U/ml and 100 µg/ml respectively; Life Technologies) and L-Glutamine (2 mM) (Life Technologies, Cergy-Pontoise, France) and either with 1% human plasma, 8% pooled human serum (pHS) or 10% fetal calf serum (FCS, Eurobio, Les Ulis, France).
HLA-A2 Monocytes were purified using centrifugal counter-flow elutriation (Clinical Transfer Facility CICBT0503, Dr. M. Grégoire, Nantes) and cultured for 4.5 days in RPMI 1640 2% human albumin in the presence of 1000 U/mL GM-CSF (Cellgenix) and 200 U/mL IL-4 (Cellgenix): MoDCs medium
Maturation of Mo-DCs was induce by addition of 1000 U/mL TNFα (Cellgenix) and 50 µg/mL poly I:C (Sigma) to the culture (Maturation MoDCs medium). Each preparation of DC was checked for purity and differentiation by flow cytometry using the markers indicated below.
Human DC phenotype was determined by the expression of CD14, CD40, CD80, CD83 and HLA-DR (data not shown).
In cross presentation assays 106 DCs were plated per well in 24 -well plates- pretreated with 3% polyHema (Sigma) for 16 hr to facilitate the harvest of DCs after the antigenic pulse.
The 10C10 clone was amplified as previously described
Synthetic long peptide Melan-A/MART-116–40 (GHGHSYTTAEELAGIGILTVILGVL) (SLP16–40) and synthetic short peptide Melan-A/MART-126–35 with the A27L modification (ELAGIGILTV), were synthesized with purity greater than 95% and purchased from Millegen (Labege, France). Synthetic fluorescent long peptides Melan-A/MART-116–40-FITC (GHGHSYTTAEELAGIGILTVILGVLK-FITC) (SLP16–40-FITC) and FITC-Melan-A/MART-116–40 (FITC-GHGHSYTTAEELAGIGILTVILGVL) (FITC-SLP16–40), were produced with purity greater than 95%. All the peptides were reconstituted at 10 mM in DMSO.
CD8 T cell activation by DCs loaded in vitro with SLP16–40 or SP26–35 peptides was determined by using APC-labeled anti-CD8+mAb and PE-labeled anti-IFNγ. Mo-DCs were stained by PKH-67 according to the manufacturer’s recommendations (Sigma) in order to exclude them from the T cell gate.
For intracytoplasmic IFNγ staining, cells were stained at 4°C for 20 minutes, with anti-CD8+ Ab. Then cells were fixed 10 minutes at room temperature in PBS 4% paraformaldehyde (Sigma). Anti-IFNγ was added to fixed cells and incubated for 30 minutes at room temperature. Reagent dilutions and washes were done with PBS containing 0.1% BSA and 0.1% saponin (Sigma). After staining, immunofluorescence was analyzed on a FACS calibur (BD Biosciences).
Alternatively, activation of the 10C10 CTL clone was evaluated by determining the IFNγ content in the supernatant in duplicates in a 16-hr CTL assay using a commercially available ELISA kit (BD Biosciences).
For the preparation of whole cell lysates, cells were lysed in a buffer containing 50 mM NaCl, 50 mM Tris, 5 mM MgCl2, 1 mM DTT, 10% glycerol and 0.1% NP40. Protein concentrations in lysates were determined using a bicinchoninic acid assay (BCA). Five to twenty µg of whole-cell lysates were separated on a 15% SDS-polyacrylamide gel and transferred onto a PVDF membrane. In some experiments 200 ng of purified 20S proteasome from erythrocytes (Standard Proteasome) or spleen (Standard Proteasome and ImmunoProteasome) were loaded as internal controls. The blots were probed with antibodies to β1i, β5i, Beta1, Beta2, Beta5, Alpha6 (all purchased from Enzo life sciences), β2i (K65/4, laboratory stock), p97/VCP (MA3-004, Dianova), Derlin-1, sec61-a and to β-actin (Santa Cruz Biotechnology) to confirm that equal amounts were present in every lane. Bound antibodies were visualized with ECL chemiluminescence (Roche).
SLP16–40 was pulsed at 10 µM, 37°C for 3 h on DCs in RPMI supplemented with 2% human albumin, 1000 U/mL GM-CSF, 200 U/mL recombinant human IL-4, 1000 U/mL TNFα and 50 µg/mL poly I:C. Following the pulse, 1.105 DC were plated per well in 96-well round-bottom plates, fixed with 0,01% glutaraldehyde containing PBS for 1 min then washed three times in RPMI. DCs were co-cultured with T-cells in the presence of 10 µg/mL Brefeldin A (BFA).
In some assays, DCs were incubated for 30 minutes before and throughout the antigen pulse period with Cytochalasine D (Sigma), ICP47 (1–35) (Millegen (Labege, France),
1.105 pulsed-DCs were co-cultured for 6 h with the 10C10 clone using 1.105 T-cells per well, in a final volume of 100 µL of RPMI containing 8% human serum and 10 µg/ml brefeldin A (Sigma, St Louis MO, USA). The DC/T cell ratio was 1∶1.
ON-TARGET plus SMART pool of interfering RNA (Dharmacon) were used to knock down p97/VCP (L-008727-00), sec61A1 (L-021503-00), Derlin-1 (E-010733-00), β1i (L-006023-00), β2i (L-006019-00) and β5i (L-006022-00). ON-TARGET plus nontargeting pool of siRNA with random nucleotides (D-001810-10) was used in each experiment as a negative control. For siRNA transfection, 4.107 cells were resuspended in 100 µl Opti-MEM without phenol red (Invitrogen) and transferred into a 4-mm electroporation cuvette (Biorad) with 1000 nmol siRNA duplex. The electroporator (Genepulser, Biorad) used a square-wave pulse of 500 V for 1 ms. Cells were then immediately transferred into 4 ml of MoDCs medium.
The mouse IgG1-mAb used in confocal microscopy were anti-HLA class-I (W6–32, produced in our laboratory), anti-GM130, anti-EEA-1, anti-LAMP-1 and anti-calreticulin (Becton Dickinson), that target respectively HLA-A,B,C and the following subcellular compartments - Golgi apparatus, early endosomes, lysosomes and ER.
DCs were pulsed for 0–48 h with SLP Melan-A16–40 FITC (10 µM) in MoDCs maturation medium. Following the pulse, DCs were fixed in a solution of 2% formaldehyde (Sigma) and stained at room temperature for 2 h either by anti-HLA or, after permeabilization in a solution of PBS containing 0.05% triton X100 and 0.05% Tween, by the mouse IgG1-mAb, anti-GM130, anti-EEA-1, anti-LAMP-1 or anti-calreticulin. Cells were washed 3 times in PBS and secondary Alexa-568 anti-mouse IgG1 (Invitrogen) was used as detection reagent. Cells were washed and nuclei were visualized with DRAQ 5 (AXXORA). Isotype control antibodies were used in all confocal microscopy experiments to confirm specificity of antibody staining. Coverslips were mount in prolong gold antifade reagent (Invitrogen) and examined with confocal microscopy. Z-series of multiple images were acquired from DCs representative of most cells in each culture.
Cell fluorescence was visualized by confocal microscopy using a NIKON A1, R, SI instrument with APO VC, X60 NA: 1,4 oil immersion objective. An argon laser at 488 nm and diode laser at 561 nm excited the fluorescence of FITC and Alexa 568 respectively; fluorescence emission was collected respectively at 525/15 for FITC and 590/15 for Alexa-568. All images were acquired at a size of 1024 pixel by 1024 pixel and had a lateral resolution of 0.21 µm by pixel. Z-step numbers and rank were chosen according to Nyquist theorem where Z = 1/3 FWHM (Full Width at Half Maximum): typically 0.25 µm for Z-range.
For analysis, Image J software was used. Colocalization volumes were calculated with the 3D object counter plugin
Quantitation of the colocalization of SLP16–40 with the different subcellular compartments (Golgi apparatus, early endosomes, lysosomes or ER) was performed by calculating the Pearson correlation coefficient, Rr, using Volocity software (Version 6.1.1 from PerkinElmer Life Sciences). An Rr value of 1 indicates complete colocalization, an Rr value of 0 indicates no specific colocalization, and an Rr value of −1 indicates a perfect but inverse correlation (exclusion). The green and red fluorescence have been determined by performing thresholding using the 3D object counter plugin default threshold method.
For the analysis of experimental data, quantitative data was compared by the Mann-Whitney U test (Graph Pad Prism 5).
In previous studies, others and we showed that the SLP15–40 or SLP16–40 are cross-presented by DC
Flow cytometry of DC activation of the 10C10 CD8+ T-cell clone 10C10. DC were pulsed for 3 h in the presence of short peptide MelanA26–35, SLP16–40, or SLP16–40-FITC before co-culture with the 10C10 clone at a 1∶1 cell ratio. The histogram represents the fluorescence emitted by the 10C10 clone stained with anti-IFN-γ.
In order to visualize the routing of SLP16–40, DCs were pulsed with SLP16–40-FITC for different times, then fixed and stained with mAbs specific for MHC class-I molecules (W6–32) and a DNA dye specific for the nucleus (Draq5). As shown in
Immunofluorescence microscopy. Kinetics of internalization of SLP16–40-FITC (green) by DCs. DCs were stained at the cell membrane with antibody w6–32 (red) and nuclei were counterstained with DRAQ 5 (blue). All image represent one single optical section. Step size of 0,5 µm thick. Original magnification, X60. Data are representative of two independent experiments. Scale bar represent a distance of 5 µm.
To define the intracellular routing, we incubated DCs for different periods of times (0 to 120 minutes) with SLP16–40-FITC and then stained DCs intracellularly with antibody to early endosomes (anti-EEA-1,
(A) Immunofluorescence microscopy. Kinetics of internalization of DCs incubated with SLP16–40-FITC (green), 15, 30, 60 or 120 minutes after pulse. DCs are stained at the early endosomes with antibody anti-EEA-1 (red) and the nuclei are counterstained with DRAQ 5 (blue). All image represent one single optical section. Step size of 0,5 µm thick. Original magnification, X60. Single scans are representative for multiple cells analysed in at least 2 experiments. (B) Immunofluorescence microscopy. Kinetics of internalization of DCs incubated with SLP16–40-FITC (green), 15, 30, 60 or 120 minutes after pulse. DCs are stained at the lysosomes with antibody LAMP-1 (red) and the nuclei are counterstained with DRAQ 5 (blue). All image represent one single optical section. Step size of 0,5 µm thick. Original magnification, X60. Single scans are representative for multiple cells analysed in at least 2 experiments. Scale bar represent a distance of 5 µm.
(A) Immunofluorescence microscopy. Kinetics of internalization of DCs incubated with SLP16–40-FITC (green), 30, 60 or 120 minutes after pulse. DCs are stained at the Golgi with antibody anti-GM130 (red) and the nuclei are counterstained with DRAQ 5 (blue). All image represent one single optical section. Step size of 0,5 µm thick. Original magnification, X60. Data are representative of two independent experiments. (B) Immunofluorescence microscopy. Kinetics of internalization of DCs incubated with SLP16–40-FITC (green), 30, 60 or 120 minutes after pulse. DCs are stained at the ER with antibody anti-calreticulin (red) and the nuclei are counterstained with DRAQ 5 (blue). All image represent one single optical section. Step size of 0,5 µm thick. Original magnification, X60. Data are representative of two independent experiments. Scale bar represent a distance of 5 µm.
To address more precisely the localization of SLP16–40 in DCs, we analyzed confocal microscopy images of each cell with the Image J 3D cell counter plugin. This application allows the measurement of the colocalization volume defined by the overlap of the green fluorescent object (SLP16–40) with the red fluorescent object (intracellular compartment) in the whole cell. To this end, we defined a threshold of no colocalization with the endoplamic reticulum staining (
(A) Volume measurement of green fluorescence (SLP16–40-FITC) with red fluorescence (ER) colocalization at each time point. For each point, average volume was determined on five different cells of two independent experiments. (B) Volume measurement of green fluorescence (SLP16–40-FITC) with red fluorescence colocalization (early endosomes or lysosomes) at each time point. Representations of colocalization in early endosomes are in white bars and colocalization in lysosomes are in black bars. For each point and each compartiment, average volume was determined in five different cells from two independent experiments. (C) The colocalization of SLP16–40-FITC immunofluorescence with intracellular compartments (early endosomes, lysosomes, ER and Golgi apparatus) was quantified by measuring the Pearson correlation coefficient (Rr) with Velocity software. A Pearson correlation of 1 indicates complete colocalization, a value of 0 indicates no specific colocalization and a value of −1 indicates a perfect but inverse correlation (exclusion). Measurements of the Pearson correlation coefficient indicate a reasonable degree of partial colocalization of SLP16–40 with early endosomes between 5 and 60 minutes and with lysosomes between 45 and 120 minutes. The Pearson correlation coefficient of SLP16–40 with ER and Golgi apparatus lysosomes indicates no specific colocalization. The Pearson correlation coefficient was measured with n = 5 cells. Statistical significance of colocalization was compared to the null hypothesis of no specific colocalization (Pearson correlation coefficient value of 0).
We evaluated the colocalization volume for early endosome and lysosome (
Quantitation of SLP16–40-FITC colocalized with early endosomes, lysosomes, ER and Golgi apparatus was measured by the Pearson correlation coefficient (Rr value). Rr values indicate specific partial colocalization of SLP16–40 with early endosomes between 5 and 60 minutes and with lysosomes between 45 and 120 minutes (
Overall, these data suggest that SLP16–40 is rapidly internalized by DCs in early endosomes, and then redirected to the lysosomes where it accumulates.
Various proteases may be involved in the processing of SLP16–40. In DCs, the proteasome is involved in generation of many MHC-I epitopes from endogenous proteins (endogenous pathway) but also from exogenous proteins during cross-presentation
(A) Untreated day 5-immature DC and DC treated with LPS (1 µg/ml) for 24 hours were analysed for their proteasome content by western-blotting using antibodies against β1, β2, β5, β1i, β2i, β5i, as indicated. Purified 26 proteasomes (250 ng) from erythrocytes (standard proteasome) and spleen (mixture of standard and immunoproteasome) were used as sources to ensure antibody specificity. To control for equal loading, proteins were subjected to western blotting using the anti-β-actin antibody. (B) Flow cytometry of DC recognition by CD8+ T-cell clone 10C10. DCs were treated with Epoxomicin (1 and 5 µM) for 30 min, then DCs were pulsed for 3 h in the presence of short peptide MelanA26–35, or SLP16–40, and inhibitor before co-culture with the 10C10 clone at a 1∶1 cell ratio. Data are representative of at least three independent experiments. Statistical analysis was performed using non-parametric Mann-Whitney test and values in the presence of inhibitor were significantly different (p<0,04).
To assess whether early cross presentation of SLP16–40 by DCs requires proteasome degradation for presentation, we used epoxomicin. This drug is a highly specific, and irreversible inhibitor of the chymotrypsin-like (CT-L), trypsin-like (T-L), and peptidyl-glutamyl peptide hydrolyzing (PGPH) activities of the proteasome which modifies the proteasomal catalytic subunits β5i, β2i, β5 and β2
To better understand the role of immunoproteasomes in this process, DC with a knockdown of any one of the three inducible subunits β1i, β2i or β5i were fed with SLP16–40 for 3 h prior to a 16-hour CTL assay in the presence of the 10C10 clone. As shown in
(A) DC were transfected with 1 µM of control siRNA or β1i, β2i, β5i -targeting siRNA for 72 hours. The knockdown of the above-stated inducible subunits as well as its impact on the steady-state level of each of the standard proteasome subunits (β1, β2, β5) was analysed by western-blotting using specific antibodies, as indicated. Antibody against b-actin was used to ensure an equal protein loading. (B) IFN-g production of the LT CD8+10C10 responded to β1i, β2i, β5i -depleted DC pulsed with either SLP16–40 or Melan- A26–35. All data are shown as means +/− SD and are representative of three independent experiments.
However, and in spite of the up-regulation of at least any of two out of three standard subunits in DC treated with a knockdown of anyone of the three inducible subunits, no major change in SLP16–40 cross-presentation could be observed (
In the cytosol, the antigens degraded into peptides are transported into the ER or ER phagosome-like compartments via the transporter associated with antigen processing (TAP) for loading onto MHC class I molecules. To investigate whether TAP was involved in the cross-presentation of SLP16–40, we used a synthetic peptide corresponding to the N-terminal 35 amino acid residues (ICP47 (1–35)), that can reach the cytosol and block TAP only in cells of DC lineage
(A) TAP transport: Flow cytometry of DC recognition by LT CD8+10C10. DCs were treated with inhibitor, ICP47 (50 and 100 µM) for 30 min, then DCs were pulsed for 3 h with the short peptide Melan-A26–35, or SLP16–40, and in the presence of the inhibitor before co-culture with the LT CD8+10C10 at a 1∶1 cell ratio. Data are representative of at least three independent experiments. Statistical analysis was performed using non-parametric Mann-Whitney’s test and values in the presence of inhibitor were significantly different (p<0.04). The molecular mechanisms involved in cross-presentation of SLP16–40. Flow cytometry of DC recognition by LT CD8+10C10. DCs were treated with inhibitor, (B) NH4Cl (20 mM and 50 mM) or (C) ExoA (10 and 20 µg/mL) for 30 min, then DCs were pulsed for 3 h in the presence of short peptide Melan-A26–35, or SLP16–40, and inhibitor before co-culture with the LT CD8+10C10 at a 1∶1 cell ratio. Data are representative of at least three independent experiments. Statistical analysis was performed using non-parametric Mann-Whitney’s test and values in the presence of inhibitor were significantly different (p<0,04).
To define more precisely the compartments involved in SLP16–40 cross-presentation by DCs, we incubated DCs for 3 hours with SLP16–40 in the presence or absence of various inhibitors. First, to elucidate the potential lysosomal involvement in cross-presentation, we used NH4Cl, an inhibitor of lysosome acidification and maturation of early endosomes into lysosomes
Following its internalization in early endosomes, translocation of SLP16–40 to the cytosol is required for subsequent degradation by cytosolic proteases and/or the proteasome
In an attempt to generate a more specific and reliable blockade of the ERAD pathway in DC, we generated knockdown DC for each of the three above-mentioned ERAD factors (i.e., p97/VCP, Derlin-1 and sec61a1). To this end, DC were treated with siRNA targeting p97/VCP, Derlin-1 or sec61a1 for three days followed by a 3-hour pulse of either SLP16–40 or Melan-A26–35 prior to a 16-hour CTL recognition assay in the presence of the 10C10 clone. As shown in
(A) Day5-immature HLA-A2+ DC were electroporated with 1 µM of either control siRNA or siRNA against p97/VCP, Derlin-1 or Sec61a1, as indicated. The steady-state protein level of each of the targeted gene was determined by western-blot analysis 72 hours later. To control for equal loading, samples were subjected to western blotting using the anti-b-actin antibody. (B) The Melan-A26–35 CTL response against siRNAtreated DC loaded with either 10 µM of SLP16–40 or 1 µM of Melan-A26–35 (as a positive control) was examined using an IFN-γ ELISA. All data are shown as means +/− SD and are representative of three independent experiments. **p<0.01 (t-Test).
The aim of this study was to characterize the SLP cross-presentation pathway in DCs. We selected for the present
Our principal results concerning SLP16–40 cross-presentation are summarized in
Here, we demonstrated, by using inhibitors and confocal microscopy that SLP16–40 is endocytosed by DCs and routed to early endosomes. The results showed that SLP16–40 was internalized very quickly after DC contact (15 to 30 minutes). Later on, specific colocalization of peptide with endosomes decreased and then disappeared from 60 minutes onwards, together with the appearance of SLP16–40 colocalization with lysosomes. The decreasing colocalization between early endosomes and SLP16–40 over time suggests an arrest of the endocytic process. One hypothesis is that DCs rapidly internalized a large quantity of SLP reaching a maximum and perhaps preventing supplemental endocytosis during the 3 h incubation with this 25-mer.
We observed a temporal correlation of colocalization between early endosomes and lysosomes (
By using cytochalasin D that inhibits actin polymerization, we prevented cross-presentation, suggesting that internalization and possibly processing of SLP16–40 require cytoskeletal actin rearrangement (data not shown). Receptor mediated endocytosis and macropinocytosis are efficient mechanisms that can guide exogenous antigens into the MHC class I and II presentation pathway in DCs
Although various mechanisms have been proposed to explain cross-presentation, the route used by internalized antigens to gain access to the cytosol for proteasomal degradation remains elusive. Over the past few years, an increasing number of a studies point to a critical role of the ER-associated degradation pathway (ERAD) in this process
However, in our hands, neither sec61 nor Derlin-1 gene silencing could significantly alter the SLP16–40 cross-presentation by DC. These findings are interesting and raise the possibility of the existence of another yet unidentified channel implicated in this process. Yet, we cannot rule out a participation of Derlin-1 and/or sec61 in other DC-based cross-presentation systems when other antigen sources are applied such as full-length proteins or cell-associated antigens including apoptotic or necrotic cells. Of note, the extraction of proteins from the ER by p97/VCP requires substrate poly-ubiquitylation. Since our SLP16–40 is a lysine-free peptide, it is conceivable that the exclusive acceptor site for poly-ubiquitylation may be represented by its N-terminus. Importantly, this implies that any blockade of the N-terminus of the SLP16–40 would prevent its cross-presentation by DC. This assumption is supported by the observation that the N-terminally FITC-modified SLP16–40 does not lead to the generation of the Melan-A26–35 peptide in DC (data not shown).
In DCs, two proteasomes exist: the standard proteasome that contains the active subunits β1, β2 and β5; and the immunoproteasome, which differs only in three active subunits (the immunosubunits β1i, β2i and β5i). Recently, the existence of additional forms of proteasomes, bearing a mixed assortment of standard and inducible catalytic subunits has been identified, which contains only one (β5i) or two (β1i and β5i) of the three inducible catalytic subunits of the immunoproteasome
This result seems to contradict previous results in which the antigenic peptide Melan-A26–35 was generated by the standard proteasome but not by the immunoproteasome
We have demonstrated the necessity of TAP for the cross-presentation of SLP16–40. The TAP transporter is expressed both in early endosomes and the ER. The use of US6 or US6 chemically linked to transferrin
Altogether, many vaccination strategies have been used to enhance antitumor responses by exploiting the cross-presentation capacities of DCs. Recently a very encouraging clinical trial for patients with SLP vaccination was published
The authors thank D. McIlroy, Dr E. Segura, and Dr. J-F. Fonteneau for carefully reading the manuscript.