The peptide GDPKHPKSF was synthesized by fully automated solid phase synthesis and Fmoc/tBu chemistry on chlorotrityl-resin. To generate the triple-chain lipopeptide, the peptide resin was elongated with the unusual amino acid Fmoc-S-(2,3-dihydroxy-2(RS)-propyl)-cysteine followed by esterification on solid phase with palmitic acid. After Fmoc-deprotection, the lipopeptide was modified by N-palmitoylation.

HEK293T cells were co-transfected with a human TLR2 plasmid and firefly luciferase under a synthetic NF-κB promoter and the constitutive Renilla luciferase reporter. Cells were stimulated with Pam₃CysSK₄ and XS15. Lysates were analysed using the Dual-Luciferase reporter assay kit (Promega, Madison, MI).

HEK-Dual hTLR2 cells (InvivoGen, San Diego, CA) were incubated with TLR1, TLR2 and TLR6 blocking antibodies (InvivoGen) or isotype control and stimuli added. SEAP levels (driven by an NF-κB promoter) were measured in supernatants (QUANTI-Blue; InvivoGen) [12].

The healthy volunteer described herein is a white male of European descent, aged 62 years at first vaccination. The individual remained healthy during the described period and reported no significant prior medical history or ongoing disease, except for preexisting arterial hypertension treated by irbesartan (150 mg) and lercanidipine hydrochloride (5 mg) as well as acetyl salicylic acid (100 mg) taken for prophylactic purposes (all medication taken once daily).

The described vaccinated individual performed all vaccinations as self-experimentation. This was undertaken voluntarily by an investigator and designer of the research himself on his own person. Interventions by physicians involved were exclusively performed after obtaining informed consent and assuring a reasonable risk-benefit assessment. In mouse toxicology studies the no-observable-effect level (NOEL) [15] was tested by administering up to 50 μg XS15, without observing any toxicity.

Since any coercion or dependency can be excluded in this case, no other party is to be protected from unethical behavior [13]. Respective conduct is widely considered appropriate and as an ethically and legally legitimate form of experimentation [13]. Self-experimentation is historically established and common among scientists, offering a route to valuable human experimentation, when performed properly [13, 14].

Anticoagulated whole blood (heparin/ citrate) or buffy coats (Center for Clinical Transfusion Medicine GmbH, Tübingen) were obtained from healthy donors after informed consent and from one vaccinated volunteer.

Peripheral blood mononuclear cells (PBMCs) were isolated by density centrifugation and either used fresh or after liquid nitrogen storage [16].

Automated peptide synthesis was performed in-house (ABI 433A; Applied Biosystems, Foster City, CA). Lyophilized peptides (see Tables 1 and 2) were diluted in DMSO or water/ DMSO for T-cell assays and monomer refolding, respectively. The former was performed by conventional refolding as described before [17, 18], whereas ADV-Hex HLA-A*01-peptide and FLU-NCAP HLA-B*08-peptide monomers were generated by exchange of an HLA-B*08 UV labile monomer [19]. Multimers were generated by incubating monomers with streptavidin-PE/ streptavidin-APC (Biolegend, San Diego, CA) together with glycerol and human serum albumin [20].Tab1

Synthetic Peptides, first vaccination and immunomonitoring

A multi-peptide vaccine was prepared mixing ADV-Hex, FLU-NCAP and EBV-GP350 peptides (Table 1) with XS15 in water/20% DMSO. This vaccine was emulgated 1:1 with Montanide™ ISA51 VG (Seppic, Paris, France) using an established protocol, injecting 400 μl, containing 80 μg XS15 and 240 μg of each peptide s.c. abdominally.

The second vaccination 14 months later contained CMV-VPAP, HLA-CMV-pp65, CMV-VIE1, CMV-pp65^283–299 and CMV-pp65^510–524 peptides (Table 2). This vaccine was prepared and administered as described, but to a different site in roughly the same lymph collection area as the first vaccination and contained 50 μg XS15 in 400 μl.

DCs were differentiated from PBMCs, culturing adherent cells with human GM-CSF and IL-4, (both PeproTech, Hamburg, Germany). Cells were either left untreated, matured with a mix of IL-1β, TNF (both PeproTech), PGE2 (Sigma-Aldrich), poly(I:C) and R848 (both InvivoGen), or treated with Pam₃CysSK₄ or XS15.

Isolation of slanMo was performed as described previously [21]. PBMCs were incubated with M-DC8 antibody containing hybridoma supernatant, labeled with rat anti-mouse IgM coupled to paramagnetic microbeads (Miltenyi Biotec, Bergisch-Gladbach, Germany) and sorted (autoMACS; Miltenyi).

CD56⁺ CD3^neg NK cells and CD3⁺ CD4⁺ T cells were isolated from PBMCs by immunomagnetic depletion (Miltenyi). Purity of sorted cells > 90% was confirmed by flow cytometry.

DCs were stained with CD14-Alexa Fluor 700 (eBioscience, San Diego, CA), CD83-APC and CD86-BV605 (Biolegend), HLA-DR-PerCP, TLR2-PE (BD Biosciences, Heidelberg, Germany) and Zombie Aqua (Biolegend) after Fc Block (BD), fixed and measured (LSR Fortessa; BD Biosciences).

Surface molecules of slanMo, NK cells, and CD4⁺ T cells were characterized with CD3-FITC, CD4-PE, CD56-PE, HLA-DR-APC (all BD), and M-DC8 hybridoma supernatant [21] to determine their purity (FACSCalibur; BD).

For intracytoplasmic staining of IFNγ and IL-4, CD4⁺ T cells were stimulated in the presence of phorbol myristate acetate (PMA) and ionomycin (both from Sigma-Aldrich) and brefeldin A was added. IFNγ-FITC and IL-4-PE (both from BD) staining was performed and analysed.

slanMo were maintained allowing spontaneous maturation into DCs and cultured in the presence of XS15 or XS15 + IFNγ to stimulate cytokine secretion. TNF, IL-1β, IL-6, IL-12, and IL-23 were determined by ELISA (BD) in supernatants. Further, matured slanMo were coincubated with the CD8⁺ T cell clone CC7 [22], in the presence of the pertinent recognized WT1 peptide RMFPNAPYL + XS15, quantifying IFNγ in supernatants. Likewise matured slanMo were coincubated with autologous NK cells and IFNγ quantified.

Matured slanMo were cocultured with allogeneic CD4⁺ T cells and XS15. Harvested T cells were incubated with PMA/ionomycin. Cells were analysed for IFNγ and IL-4 production by flow cytometry.

Results were assessed by Student’s t-test or Analysis of Variance (ANOVA), with p ≤ 0.05 considered significant.

Fresh PBMCs were cultured either alone, with Pam₃CysSK₄ or XS15, or a mix of phytohaemagglutinin-L (PHA) and Pokeweed mitogen (PWM). Adherent and non-adherent cells were stained with mAbs: CD3-BV711, CD56-BV421, CD19-BV785, Zombie aqua (all Biolegend), CD14-Alexa Fluor 700, HLA-DR-PerCP, and CD69-APC-Cy7 (all BD). Cells were measured by flow cytometry as described above.

After surgical removal of the vaccine-induced granuloma, tissue was used for in vitro expansion of granuloma infiltrating T cells (GICs) after dissociation by combined mechanical and enzymatic processes, filtering (100 μm) and separation over a density gradient. Isolated cells were phenotyped and measured (ELISpot assay).

GICs and PBMCs were stained for CD3-PE-Cy5.5 (eBioscience), CD4-BV711, CD8-PerCP, CD25-BV605, CD45RA-BV570, CCR7-BV650, CD39-BV421, PD-1-APC-Cy, BTLA-PE, Tim-3-PE-Cy7 (all Biolegend), LAG3 (Enzo Life Sciences, Lörrach, Germany), CTLA4-PE-CF594 (BD) and Live/dead-Aqua dye (Life technologies, Carlsbad, CA) or with isotype controls. Cells were fixed and permeabilized, followed by ICS using Foxp3-FITC (eBioscience) and Ki67-Alexa Fluor 700 (BD) and measured on an LSR Fortessa (BD).

Granuloma tissue pieces were cultured and expanded for 12 days in specialized TIL culture medium containing IL-2 and anti-CD3 antibody (clone OKT3, Miltenyi).

IFNγ secretion by PBMCs and GICs in response to peptide stimulation was determined using ELISpot assay [23].

Multimer staining conformed essentially to the protocol suggested by CIP (http://www.cimt.eu/workgroups/cip) as outlined previously [6].

For a rough estimate of vaccine-specific T cells, respective cells within the granuloma were calculated based on experimental results (see Additional file 1: Supplementary Materials and Methods).

ICS was performed as reported earlier [23]. Cells were stimulated with individual peptides or with an equal volume of water/ 10% DMSO in the presence of anti-CD107a (BD), GolgiStop (BD) and Brefeldin A (Sigma-Aldrich). After 12 h cells were stained for CD4-APC-Cy7 (BD), CD8-PECy7 (Beckman Coulter) and CD3-BV711 (Biolegend) and with Aqua Live Dead, fixed and permeabilized in (Cytoperm/Cytofix; BD) and further stained for IFNγ-Alexa Fluor 700 (BD Biosciences), anti-TNF-Pacific Blue (Biolegend), IL-10-PE and IL-2-APC (both BD).

Levels of 42 proteins and immune-associated markers were measured using the Luminex 100/200 instrument. The kit components and software for data analysis of the multiplexed immunoassay were kindly provided by Myriad RBM, Austin, TX (http://rbm.myriad.com) and used as specified. Serum samples were tested in singles.

Antibodies were detected by ELISA with an in-house assay as published previously [24]. XS15 coated microtiter-plates were incubated with sera from the vaccinated individual as well as pertinent controls. Bound antibodies were detected with peroxidase conjugated goat anti-human IgG- and IgM-antibodies (DIANOVA, Hamburg, Germany).

HLA class I and HLA-DR ligands were isolated by immunoaffinity purification from granuloma tissue with W6/32 and L243 antibodies (both produced in-house) as described previously [25]. HLA ligand extracts were analysed by tandem mass spectrometry (LC-MS/MS) using an Orbitrap Fusion Lumos and Ultimate3000 RSLCnano system (both ThermoFisher Scientific). Data processing was performed by SEQUEST database search against the reviewed Swiss-Prot human reference proteome concatenated with the vaccinated peptide sequences, verifying identifications by comparison with fragmentation patterns of isotope-labeled peptides of identical sequence.

RNASeq was performed by an external service provider (CeGaT, Tübingen, Germany). RNA was isolated from the granuloma center & margin and distal edge. Single end sequencing was performed (HiSeq 2500; Illumina San Diego CA). Mapping (hg19) (STAR software, V. 2.4.0), data processing and counts of mapped reads were computed (Cufflinks Tool Suite; Version 2.1.1). FPKM values were calculated (Cuffdiff) employing a pooled-variance model and geometric normalization with multi-read-correction (Additional files 2, 3, 4). Differential gene expression (FC > 5, q < 0.05) in the granuloma center vs. the margin was assessed (Additional file 5) and a pre-selected gene set of interest (hallmark inflammatory response gene set, comprising 200 genes; last accessed: December 2018; http://software.broadinstitute.org/gsea/msigdb/cards/HALLMARK_INFLAMMATORY_RESPONSE.html) compared for the three different tissue regions sampled (Additional file 6).

A tissue sample of the granuloma center was processed as formalin-fixed paraffin embedded (FFPE) tissue, cut into 3–5 μm sections and stained by HE for histological evaluation. Granulocytes were identified by typical appearance as well as mineral oil deposits (representing vaccine remnants) appearing as large vacuolar structures. Immunohistochemistry staining was performed (BOND-MAX, Leica Biosystems, Wetzlar, Germany) with monoclonal antibodies recognizing CD8, CD68, CD20 (all Dako, Glostrup Denmark) and CD4 (Cell Marque, Rocklin, CA). Appropriate positive and negative controls were included.

FFPE tissue sections were deparaffinized in xylene, hydrated by washes of graded ethanol to water and boiled in citrate buffer. Tissues sections were stained with mouse anti-CD8 antibodies (Dako) and the mouse anti-slan antibody DD2 (in-house, Institute of Immunology, Medical Faculty Carl Gustav Carus, Dresden). CD8⁺ T cells were visualized by an AF633-labeled goat anti-mouse IgG antibody (ThermoFisher Scientific) and slanMo by a goat anti-mouse IgM Biotin (1:100, Southern Biotech, Birmingham, AL), followed by AF546-labeled Streptavidin (ThermoFisher Scientific). Tissues were mounted on DAPI-containing AKLIDES® ANA plus medium (Medipan, Dahlewitz, Germany), coverslipped and evaluated (BZ-9000; Keyence, Osaka, Japan). For quantification of slanMo and CD8⁺ T cells, positively stained cells were counted in 15 different high power fields (HPF) of a tissue section using the Vectra imaging platform (Akoya Biosciences, Hopkinton, MA, USA) and the mean value was determined. The mean number of cells per HPF (area: 0.3345mm²) was converted to square millimeter.

To investigate the injection site and draining lymphoid organs, a dynamic positron-emission tomography (PET)/ magnetic resonance tomography (MR) scan of the abdomen was performed after injection of 209 MBq ¹⁸F-2-Fluor-2-desoxy-D-glucose (¹⁸F-FDG; i.v.) using a 3 T-PET/MR scanner (Biograph mMR, Siemens Healthineers, Erlangen, Germany). PET was reconstructed with an OSEM-3D algorithm, applying a MR-based attenuation map. For morphologic analysis, a T2 Half-Fourier Acquisition Single-shot Turbo spin Echo (HASTE) and a T2 Turbo inversion recovery magnitude TIRM sequence was assessed.

Pam₃Cys-derivates, such as Pam₃Cys-SK4 [26], are water-soluble amphiphilic compounds, exhibiting detergent characteristics and can induce unspecific effects at higher concentrations [27]. We therefore designed a new lipopeptide (chemical structure in Fig. 1a) with nearly even charge balance, derived from a naturally occurring sequence (GDPKHPKSF) in Mycoplasma salivarium [28]. The compound can be generated in very high purity by conventional chemistry and purification procedures, is water-soluble, can be sterilized by 0.2 μm filtration, and thus is GMP-amenable. This new compound was designated XS15.Fig1Fig. 1

Pam₃Cys-GDPKHPKSF (XS15) is a TLR1/2 ligand activating immune cells and stimulating DCs and cytokine release. (a) Structure of Pam₃Cys-GDPKHPKSF: Skeletal structural formula of the molecular structure of the lipopeptide Pam₃Cys-GDPKHPKSF termed XS15. (b) Dual-luciferase assay on HEK293T cells transfected with TLR2: HEK293T cells were transiently transfected with a human TLR2 plasmid and a NF-κB luciferase reporter plasmid or left untreated (− ctrl.). Culture medium was replaced after 30 h and stimuli added at the stated concentrations. The cells were incubated for 18 h and lysates were prepared and analysed by dual-luciferase assay. Pam₃CysSK₄ (P3CSK4) and two different lots of XS15 (XS15#1/ XS15#2) were used. (c) HEK-Dual hTLR2 cells, stably expressing a NF-κB/AP-1-inducible secreted embryonic alkaline phosphatase (SEAP) reporter, were incubated for 1 h with TLR1, TLR2 and TLR6 blocking antibodies, isotype control or negative controls (no Abs) (4 μg/ml). Then, cells were stimulated for 24 h with the established TLR2/6 agonist FSL-1 (1 ng/ml), XS15 (10 ng/ml) or left unstimulated (− ctrl.). Supernatants were collected and SEAP levels determined using QUANTI-Blue detection assay. Error bars represent SD. The graph shows the mean + SEM of n = 2 experiments, significance was assessed by two-way ANOVA. (d) Immune cell activation by XS15: Fresh PBMCs were cultured for 40 h in the presence of Phytohemagglutinin-L (PHA) + Pokeweed (PWM) (P + P), Pam₃CysSK₄ (P3CSK4), XS15 or left untreated (− ctrl.). Activated NK (left panel) and B cells (right panel) were assessed with the marker CD69 following the gating strategy: time gate, single cells (FSC-H/ FSC-A), living cells (Zombie-Aqua/ FSC-A), lymphocytes (FSC-A/ SSC-A); B-cells were defined as CD14^neg CD3^neg CD19⁺ cells and NK cells as CD14^neg CD3^neg CD19^neg CD56⁺ cells. Healthy donors (n = 6), means are shown, significance was assessed by one-way ANOVA. (e) Dendritic cell (DC) stimulation by XS15: DCs were differentiated from blood monocytes and then matured as described in the material and methods section. Gating strategy was: time gate, single cells (FSC-H/ FSC-A), living cells (Zombie Aqua/ FSC-A). Upper panel: scatter plots for healthy donors (n = 6), means are shown significance was assessed by one-way ANOVA. Lower panel: modal histograms and median fluorescences for one representative donor. Medium control without maturation cocktail = − ctrl. Standard maturation cocktail = Mat. (f) Induction of cytokine release by XS15: Anticoagulated whole blood was incubated with XS15 (10 μg/ml) as well as LPS (100 ng/ml) and PHA (2 μg/ml)/ PWM (1 μg/ml) as positive (+ ctrl.) and medium only as negative controls (− ctrl.) and supernatants harvested after 20 h. Multiplexed bead-based sandwich immunoassays were performed using a LUMINEX device with a 42-analyte panel. Exemplary findings obtained in three healthy donors (HD) for IL-8 (left), MCP1 (middle) and MIP-1β (right) are shown with means. HD1 (blue square) designates the vaccinated volunteer characterized in more detail subsequently. Additional results are provided in Additional file 7: Table S1. In case of saturation, the upper limit of quantification (ULOQ) was assigned. p ≤ 0.05*; **p ≤ 0.01; ***p ≤ 0.001

To confirm TLR2 activity, we used HEK cells transiently transfected with TLR2 in an NF-κB reporter system, as established readout to measure TLR2 activity [29]. Dose escalations compared to the standard Pam₃CysSK₄ revealed a similar activity of XS15, absent in TLR2-negative HEK cells (Fig. 1b). Since it is established that Pam₃Cys is a ligand of TLR1/2 heterodimers, also by crystal structure analysis [30], we assumed that XS15 is also a TLR1/2 ligand. This was confirmed by antibody blocking experiments (Fig. 1c). Incubation of PBMCs with XS15 showed CD69 induction on B (p = 0.055), but not on NK cells, within 40 h (Fig. 1d), both cell types were reported to show similar TLR2 levels, whereas B cells show increased TLR1 expression [31]. The stimulation of monocyte-derived DCs with XS15 significantly induced HLA-DR, CD83 and CD86, in line with the reported expression of TLR2 on DCs [32] (Fig. 1e). To assess induction of cytokine production, fresh citrate anticoagulated whole blood from three volunteers was incubated with XS15, LPS or PHA/ PWM as positive control. After 20 h, supernatant was harvested and subjected to Luminex multiplexed bead-based sandwich immunoassays. A particularly strong induction of IL-8, MCP1, and MIP-1β was observed, albeit with considerable inter-donor variance as commonly observed in humans [33], indicative of the activation of innate immune cells (Fig. 1f; Additional file 7: Table S1).

6-sulfo LacNAc⁺ monocytes (slanMo, formerly termed M-DC8⁺ DCs or slanDCs) represent a particularly proinflammatory subset of human non-classical blood monocytes that can undergo a differentiation process into DCs [21, 34–36]. Previously, we have demonstrated that slanMo display prominent expression of TLR2 and produce large amounts of various proinflammatory cytokines upon activation with TLR2 agonists [21, 34]. Further studies revealed that slanMo efficiently activate T lymphocytes and NK cells [21, 36, 37]. Based on these proinflammatory features of slanMo, we explored the impact of XS15 on various immunostimulatory properties of this cell subset. To investigate the influence of XS15 on their cytokine release, slanMo were maintained for 6 h to allow spontaneous maturation into DCs and cultured in the presence of XS15 subsequently. XS15 efficiently enhanced the capacity of slanMo to secrete the proinflammatory cytokines TNF, IL-1β, IL-6, and IL-23 (Fig. 2a), whereas IL-12 production was not influenced. Interestingly, combined XS15 and IFNγ significantly augmented IL-12 release by slanMo (Fig. 2b).Fig2Fig. 2

Impact of XS15 on cytokine release by slanMo and their capacity to stimulate WT1 peptide-specific CD8⁺ T cells and NK cells. (a) slanMo were maintained for 6 h to allow spontaneous maturation into DCs. Subsequently, slanMo were cultivated alone (slanMo) or in the presence of XS15 (slanMo + XS15) for additional 18 h. Supernatants were collected and the concentration of (a) TNF (left), IL-1β (middle), IL-6 (right), IL-23 (lower left) analysed by ELISA. (b) slanMo were maintained for 6 h to allow spontaneous maturation into DCs. Subsequently, slanMo were cultivated in the absence (slanMo) or presence of XS15 (slanMo + XS15) for additional 18 h, alternatively, slanMo were incubated with IFNγ for the first 6 h. Thereafter, slanMo were cultivated in the presence of IFNγ alone (slanMo + IFNγ) or together with XS15 (slanMo + IFNγ + XS15) for additional 18 h. Then, IL-12 was analysed by ELISA. The results of three different healthy donors (HD) are presented as mean ± SE of duplicate or triplicate measurements. (c) Effect of XS15 on the capacity of slanMo to stimulate IFNγ release by WT1 peptide-specific CD8⁺ T cells: slanMo were maintained for 6 h to allow spontaneous maturation. Subsequently, slanMo were coincubated with the specific CD8⁺ T cell clone CC7 (slanMo + CD8⁺), in the presence of the WT1 peptide (WT1) and/or XS15. After 42 h, supernatants were collected and IFNγ was quantified by ELISA. The results of three different healthy donors (HD) are presented as mean ± SE of triplicate determinations. (d) Impact of XS15 on the ability of slanMo to stimulate IFNγ secretion by NK cells: slanMo were maintained for 6 h to allow spontaneous maturation. Then, autologous NK cells were cultured either alone (NK) or incubated with XS15 (NK + XS15), cocultured with slanMo alone (NK + slanMo) or additionally incubated with XS15 (NK + slanMo +XS15). After 42 h, supernatants were collected and the concentration of IFNγ was determined by ELISA. The results of three different HD are presented as mean ± SE of triplicate determinations. Asterisks indicate a statistically significant difference (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; assessed by Student’s t-test). Exemplary flow cytometry results showing effects of XS15 on slanMo-mediated T cell programming regarding the percentage of IFNγ- and IL-4-producing CD4⁺ T cells are provided as Additional file 8: Fig. S1

We assessed whether XS15 might prove as an effective adjuvant with properties similar to CpG, when used combined with Montanide [6, 39]. An HLA-A*01-restricted adenovirus-derived 10 amino acid (AA) peptide (ADV-Hex, LTDLGQNLLY), an HLA-B*08 influenza-derived 9 AA peptide (FLU-NCAP, ELRSRYWAI) and a promiscuous HLA-DR restricted 15 AA EBV peptide (EBV-GP350, PRPVSRFLGNNSILY), dosed at 240 μg/peptide (Table 1), were emulsified in Montanide together with 80 μg of XS15 and injected subcutaneously (s.c.; 400 μl) in the lower abdomen of an HLA-matched volunteer. A time line depicting the course of events is provided in Fig. 3a. Ex vivo IFNγ ELISpot assays (300,000 PBMCs/well) obtained at days 28 and 44 after vaccine administration showed strong reactivities against the HLA class I (107–208 spots) and the HLA class II (416–726 spots) peptides (Fig. 3b). Pre-vaccination ELISpots were negative for the HLA class II peptide and weak for both class I peptides (8–24 spots). Such a strong induction of human T cells in vivo has never been evidenced by us before as a result of any other treatment and is therefore unprecedented in our laboratory (Fig. 3c), however it should be noted that a single case report is unable to provide any conclusive evidence. In a vaccination study in prostate carcinoma patients, utilizing peptides emulsified in Montanide with or without additional adjuvants, we did not detect any ex vivo ELISpot responses, not even after four repetitive vaccinations ( [23, 40]; and unpublished own data). In a study in renal cell carcinoma patients using multi-peptide vaccination (i.d.) and GM-CSF, T cell responses against viral or tumor antigens could only be detected after in vitro restimulation [5]. Since ex vivo ELISpot is considered to reflect the activity and quantity of effector T cells, we conclude that the massive induction of functional T cells in this volunteer is best explained by the peptide vaccination with XS15. The individual’s serum was also tested for antibody responses against the vaccine components (days 28, 44, 70, and 119 after the first vaccination). A vigorous induction of antibodies against the vaccine peptides was not observed. Only a moderate IgM induction, but no other antibody class, was observed against XS15 and/or the attached peptide GDPKHPKSF (Additional File 7: Table S2).Fig3Fig. 3

A single vaccination with peptides and XS15 induces a granuloma and local immune cell infiltration with functional T cells. (a) Time line providing an overview on blood and tissues samples as well as analyses described subsequently and interventions performed (i.e. vaccination, ¹⁸F-FDG-PET-MR imaging/ granuloma resection). Vaccinated peptides used at each time point are provided in Tables 1 & 2, respectively; Pre (before vaccination); d (day after first vaccination). (b) Induction of functional T cells by XS15 detected in ex vivo ELISpot: PBMCs were isolated from peripheral blood of a volunteer before vaccination (pre-vac), 28 days and 44 days after vaccination. IFNγ response towards the three vaccine peptides (ADV-Hex, FLU-NCAP and EBV-GP350) was determined in two ELISpot assays (Pre-vac + 28d, and 44d). HIV-A*01, HIV-B*08 and Fil-A peptides served as the relevant negative controls. 300,000 cells were seeded per well. Phytohemagglutinin-L (PHA-L) stimulation was used as a positive control (ELISpot plate wells rearranged, and negative controls left out). (c) Respective mean spot counts and SD/100,000 cells per well are shown. (d) Granuloma formation at the vaccination site: ¹⁸F-FDG-PET/MR (upper panel) performed on day 43 demonstrated an intense ¹⁸F-FDG uptake at the site of the induration (standardized uptake value ((SUV(mean) 4.6; SUV(max) 6.4), but no ¹⁸F-FDG-uptake was observed in the draining lymph nodes or in any other secondary lymphoid organs; corresponding MR (lower panel). (e) Immune cell infiltration of the granuloma induced by vaccination: A tissue sample from the granuloma center was processed as formalin-fixed paraffin-embedded (FFPE) tissue and assessed by hematoxylin & eosin (HE) staining (right) and immunohistochemistry (left). T cells (CD8⁺ and CD4⁺), B cells (CD20⁺) as well as macrophages (CD68⁺) and granulocytes appeared as ordered structures in separated areas resembling lymphoid tissues. Mineral oil deposits (black arrows) were still discernible, surrounded by macrophages, whereas both CD4⁺ and CD8⁺ T cells were located closely to the macrophages but separated from the oil patches. Original magnification was × 100. Black scale bars indicate 200 μm. (f) Co-localization of slanMo and CD8⁺ T cells in the granuloma. Immunofluorescence staining was performed to detect slanMo and CD8⁺ lymphocytes in the granuloma of the XS15-vaccinated volunteer. As representative examples, images of single CD8⁺ T cell or slanMo stainings as well as merged images are shown. Original magnification was × 400. White scale bars are 20 μm

As expectable with Montanide, a painless granuloma formed at the injection site. The volume increased to about 8 ml, as measured by ultrasound (days 17 and 41), without any sonographic signs of infection. After 21 days it appeared as a well-palpable induration of about 2 × 2 cm, with a central reddish surface. The granuloma was described as not touch-sensitive, whereas the skin surface as slightly itching. Since the PBMCs showed a strong and functional T cell response at day 28, we assessed its metabolic activity and performed a simultaneous PET/MR scan after injection of the glucose analogue ¹⁸F-2-Fluor-2-desoxy-D-glucose (¹⁸F-FDG) on day 43. An intense ¹⁸F-FDG uptake at the site of the granuloma was observed (standardized uptake value (SUV(mean)) 4.6; SUV(max) 6.4), obviously caused by the massive inflammatory response (Fig. 3d). No ¹⁸F-FDG-uptake was observed in the draining lymph nodes or any other secondary lymphoid organs. Since granulomas caused by Montanide with or without adjuvant may sequester T cells and induce their dysfunction and deletion in mice [7], we aimed to test whether this is reproduced in humans, and therefore surgically removed the granuloma at day 44. FFPE tissue samples from the granuloma center showed T cells (CD8⁺ and CD4⁺) as well as macrophages (CD68⁺), B cells (CD20⁺) and granulocytes appearing as ordered structures in separated areas, resembling lymphoid tissues. Mineral oil deposits (Fig. 3e, black arrows) were still discernible, surrounded by macrophages, whereas both CD4⁺ and CD8⁺ T cells were located closely to macrophages but separated from the oil patches. In line with our findings that XS15 efficiently enhances important immunostimulatory properties of slanMo, granuloma-infiltrating slanMo were detectable (18.9 slanMo/mm²) and can co-localize with CD8⁺ T lymphocytes (461.8 CD8⁺ T cells/mm²) as demonstrated in (Fig. 3f).

A single cell suspension was prepared from fresh tissue in the center of the granuloma. The GICs consisted of B, T and NK cells, monocytes and granulocytes. Both CD8⁺ and CD4⁺ T cells expressed activation markers (CD25) and proliferated (intracellular Ki67). The majority was of the effector memory phenotype, with much higher frequencies than in PBMCs obtained at the same day (Additional File 8: Fig. S3). The frequency of regulatory T cells (T_reg; Foxp3⁺CD25⁺) among CD4⁺ cells was similar in the PBMCs vs. GICs (approx. 11%) (Additional file 8: Fig. S4), in addition different checkpoint receptors were characterized in both cell subsets (Additional File 8: Fig. S5). Ex vivo IFNγ ELISpot of GICs (/50.000 cells) revealed an average of 152 and 125 specific spot counts for the HLA class I peptides (ADV-Hex and FLU-NCAP, respectively) and 568 spots for the HLA class II peptide (GP350), with a background of approx. 32 spots, likely due to remnant vaccine peptides on antigen presenting cells within the granuloma (Fig. 4a). This notion was supported by mass spectrometric detection of all vaccinated peptides in HLA ligand extracts purified from the granuloma core (Additional File 8: Fig. S6). Vaccine-specific T cells among GICs and PBMCs were stained by relevant HLA class I peptide-MHC multimers (Fig. 4b); moreover, they were characterized to be multifunctional after in vitro expansion, confirmed by production of IFNγ, TNF, IL-2 and CD107a, but not IL-10 (Fig. 4c). The total number of vaccine-antigen specific functional T cells was estimated at 3.0 × 10⁵ in the granuloma and 20.5 × 10⁶ in peripheral blood. Thus, in contrast to data reported from mice [7], the granuloma evidenced in a human volunteer induced by Montanide, peptide and XS15 did not show features of a destructive sink for the majority of antigen specific T cells.Fig4Fig. 4

Functionality and antigen-specificity of granuloma infiltrating cells (GICs). GICs were isolated as described in the Material and Methods section and analysed alongside PBMCs isolated from blood drawn on the same day from the same individual. (a) GICs were rested overnight after isolation and the IFNγ response towards the three vaccinated peptides (ADV-Hex, FLU-NCAP and EBV-GP350; Table 1) was determined by IFNγ ELISpot assay. 50,000 cells were seeded per well. HIV-A*01, HIV-B*08 and Fil-A peptides served as the relevant negative controls (rearranged wells). Ex vivo phenotype of GICs is provided as Additional File 8: Fig. S3. (b) PBMCs and GICs were harvested from the ELISpot plate (see panel A) and stained with ADV-Hex APC- and FLU-NCAP-PE- multimers. Percentages of CD8⁺ multimer-positive and multimer-negative cells within CD4^neg are indicated. (c) GICs were stimulated and expanded in vitro using anti-CD3 mAb and IL-2. The cells were then re-stimulated with the indicated peptides or with an equal volume of 10% DMSO for 12 h and the indicated secreted cytokines and surface CD107a expression (degranulation) were quantified by flow cytometry (% of functional cells are given after subtraction of marker-positive cells in the DMSO control well)

More than one year after the first vaccination, the volunteer (CMV seronegative), was vaccinated with a new multi-peptide cocktail (Tab. 2). The vaccine contained five CMV-derived peptides as well as the EBV-GP350 peptide used already for the first vaccination, now combined with 50 μg of XS15. The HLA class I peptides induced a weak ex vivo T cell response (Fig. 5a; top panel), which increased after a short in vitro pre-sensitization with the respective peptides (Fig. 5a; middle panel). Reactivity against the EBV-GP350 peptide, which had been used in the first vaccination 14 months before, was still detectable ex vivo (approx. 60 spots) before the second vaccination, and increased to more than 900 spots one month after the second vaccination (Fig. 5b), indicating a strong boosting effect. Both newly vaccinated HLA class II CMV peptides stimulated a strong ex vivo T cell response after one single vaccination.Fig5Fig. 5

Induction of CMV specific T cells after a single multi-peptide vaccination, and evidence for long lasting memory and boosting. The same volunteer as previously shown was vaccinated with the peptides shown in Table 2, this time with 50 μg of a new batch of XS15. At day 28 after vaccination (Post-vac), PBMCs were assayed by ex vivo ELISpot (a; upper panel and b, 300.000 cells/well), and additionally tested after a short time of in vitro expansion in the presence of the relevant peptides (in vitro stimulation; IVS) (A; lower panels, 250.000 cells/well). Reactivities against the HLA class I and HLA class II peptides are shown in panels (a) and (b), respectively (rearranged wells). In addition, bar charts with respective mean spot counts /100,000 cells + SD (when applicable) are shown. Negative control (− ctrl.) was DMSO or respective HLA-matched peptides (HIV); vac (vaccination)