PD-1-cis IL-2R agonism yields better effectors from stem-like CD8+ T cells.

Expansion and differentiation of antigen-experienced PD-1+TCF-1+ stem-like CD8+ T cells into effector cells is critical for the success of immunotherapies based on PD-1 blockade1-4. Hashimoto et al. have shown that, in chronic infections, administration of the cytokine interleukin (IL)-2 triggers an alternative differentiation path of stem-like T cells towards a distinct population of 'better effector' CD8+ T cells similar to those generated in an acute infection5. IL-2 binding to the IL-2 receptor α-chain (CD25) was essential in triggering this alternative differentiation path and expanding better effectors with distinct transcriptional and epigenetic profiles. However, constitutive expression of CD25 on regulatory T cells and some endothelial cells also contributes to unwanted systemic effects from IL-2 therapy. Therefore, engineered IL-2 receptor ß- and γ-chain (IL-2Rßγ)-biased agonists are currently being developed6-10. Here we show that IL-2Rßγ-biased agonists are unable to preferentially expand better effector T cells in cancer models and describe PD1-IL2v, a new immunocytokine that overcomes the need for CD25 binding by docking in cis to PD-1. Cis binding of PD1-IL2v to PD-1 and IL-2Rßγ on the same cell recovers the ability to differentiate stem-like CD8+ T cells into better effectors in the absence of CD25 binding in both chronic infection and cancer models and provides superior efficacy. By contrast, PD-1- or PD-L1-blocking antibodies alone, or their combination with clinically relevant doses of non-PD-1-targeted IL2v, cannot expand this unique subset of better effector T cells and instead lead to the accumulation of terminally differentiated, exhausted T cells. These findings provide the basis for the development of a new generation of PD-1 cis-targeted IL-2R agonists with enhanced therapeutic potential for the treatment of cancer and chronic infections.

Photochemically-Mediated Inflammation and Cross-Presentation of Mycobacterium bovis BCG Proteins Stimulates Strong CD4 and CD8 T-Cell Responses in Mice.

Conventional vaccines are very efficient in the prevention of bacterial infections caused by extracellular pathogens due to effective stimulation of pathogen-specific antibodies. In contrast, considering that intracellular surveillance by antibodies is not possible, they are typically less effective in preventing or treating infections caused by intracellular pathogens such as Mycobacterium tuberculosis. The objective of the current study was to use so-called photochemical internalization (PCI) to deliver a live bacterial vaccine to the cytosol of antigen-presenting cells (APCs) for the purpose of stimulating major histocompatibility complex (MHC) I-restricted CD8 T-cell responses. For this purpose, Mycobacterium bovis BCG (BCG) was combined with the photosensitiser tetraphenyl chlorine disulfonate (TPCS2a) and injected intradermally into mice. TPCS2a was then activated by illumination of the injection site with light of defined energy. Antigen-specific CD4 and CD8 T-cell responses were monitored in blood, spleen, and lymph nodes at different time points thereafter using flow cytometry, ELISA and ELISPOT. Finally, APCs were infected and PCI-treated in vitro for analysis of their activation of T cells in vitro or in vivo after autologous vaccination of mice. Combination of BCG with PCI induced stronger BCG-specific CD4 and CD8 T-cell responses than treatment with BCG only or with BCG and TPCS2a without light. The overall T-cell responses were multifunctional as characterized by the production of IFN-γ, TNF-α, IL-2 and IL-17. Importantly, PCI induced cross-presentation of BCG proteins for stimulation of antigen-specific CD8 T-cells that were particularly producing IFN-γ and TNF-α. PCI further facilitated antigen presentation by causing up-regulation of MHC and co-stimulatory proteins on the surface of APCs as well as their production of TNF-α and IL-1ß in vivo. Furthermore, PCI-based vaccination also caused local inflammation at the site of vaccination, showing strong infiltration of immune cells, which could contribute to the stimulation of antigen-specific immune responses. This study is the first to demonstrate that a live microbial vaccine can be combined with a photochemical compound and light for cross presentation of antigens to CD8 T cells. Moreover, the results revealed that PCI treatment strongly improved the immunogenicity of M. bovis BCG.

Photochemical internalization (PCI)-mediated activation of CD8 T cells involves antigen uptake and CCR7-mediated transport by migratory dendritic cells to draining lymph nodes.

Antigen cross-presentation to cytotoxic CD8+ T cells is crucial for the induction of anti-tumor and anti-viral immune responses. Recently, co-encapsulation of photosensitizers and antigens into microspheres and subsequent photochemical internalization (PCI) of antigens in antigen presenting cells has emerged as a promising new strategy for inducing antigen-specific CD8+ T cell responses in vitro and in vivo. However, the exact cellular mechanisms have hardly been investigated in vivo, i.e., which cell types take up antigen-loaded microspheres at the site of injection, or in which secondary lymphoid organ does T cell priming occur? We used spray-dried poly(lactic-co-glycolic acid) (PLGA) microspheres loaded with ovalbumin and the photosensitizer tetraphenyl chlorine disulfonate (TPCS2a) to investigate these processes in vivo. Intravital microscopy and flow cytometric analysis of the murine ear skin revealed that dendritic cells (DCs) take up PLGA microspheres in peripheral tissues. Illumination then caused photoactivation of TPCS2a and induced local tissue inflammation that enhanced CCR7-dependent migration of microsphere-containing DCs to tissue-draining lymph nodes (LNs), i.e., the site of CD8+ T cell priming. The results contribute to a better understanding of the functional mechanism of PCI-mediated vaccination and highlight the importance of an active transport of vaccine microspheres by antigen presenting cells to draining LNs.

Blockade of programmed cell death protein 1 (PD-1) in Sézary syndrome reduces Th2 phenotype of non-tumoral T lymphocytes but may enhance tumor proliferation.

Sézary syndrome (SS) is an aggressive leukemic variant of cutaneous T-cell lymphoma (L-CTCL) that arises from malignant clonally derived skin-homing CD4+ T cells. Based on advancements in our understanding of the mechanisms underlying L-CTCL, boosting the suppressed immune response emerges as a promising strategy in SS management. Immune checkpoint inhibitory molecules have already demonstrated efficacy in a wide spectrum of malignancies. Currently, agents targeting the programmed death-1 (PD-1) axis are under evaluation in L-CTCL. Here we investigated the expression of PD-1 and its ligands, PD-L1 and PD-L2 in blood and skin from patients with L-CTCL. We demonstrate that PD-1 expression is markedly increased on tumor T cells compared to non-tumor CD4+ T cells from SS patients and to CD4+ cells from healthy individuals. In contrast, PD-L1 shows decreased expression on tumor T cells, while PD-L2 expression is low without significant differences between these groups. Functional PD-1 blockade in vitro resulted in reduced Th2 phenotype of non-tumor T lymphocytes, but enhanced the proliferation of tumor T cells from SS patients. Our study sheds some light on the PD-1 axis in both peripheral blood and skin compartments in SS patients, which may be relevant for the treatment of L-CTCL with immune checkpoint inhibitor.

Combined Photosensitization and Vaccination Enable CD8 T-Cell Immunity and Tumor Suppression Independent of CD4 T-Cell Help.

Cytotoxic T lymphocytes (CTLs) are key players in fighting cancer, and their induction is a major focus in the design of therapeutic vaccines. Yet, therapeutic vaccine efficacy is limited, in part due to the suboptimal vaccine processing by antigen-presenting cells (APCs). Such processing typically takes place via the MHC class II pathway for CD4 T-cell activation and MHC class I pathway for activation of CD8 CTLs. We show that a combination of skin photochemical treatment and immunization, so-called photochemical internalization (PCI) facilitated CTL activation due to the photochemical adjuvant effect induced by photosensitizer, oxygen, and light. Mice were immunized intradermally with antigen and photosensitizer, followed by controlled light exposure. PCI-treated mice showed strong activation of CD8 T cells, with improved IFN-γ production and cytotoxicity, as compared to mice immunized without parallel PCI treatment. Surprisingly, the CD8 T-cell effector functions were not impaired in MHC class II- or CD4 T-cell-deficient mice. Moreover, PCI-based vaccination caused tumor regression independent of MHC class II or CD4 T cells presence in melanoma bearing mice. Together, the data demonstrate that PCI can act as a powerful adjuvant in cancer vaccines, even in hosts with impaired T-helper functions.

Cationic Liposomes: A Flexible Vaccine Delivery System for Physicochemically Diverse Antigenic Peptides.

PURPOSE: Personalized peptide-based cancer vaccines will be composed of multiple patient specific synthetic long peptides (SLPs) which may have various physicochemical properties. To formulate such SLPs, a flexible vaccine delivery system is required. We studied whether cationic liposomes are suitable for this purpose. METHODS: Fifteen SIINFEKL T cell epitope-containing SLPs, widely differing in hydrophobicity and isoelectric point, were separately loaded in cationic liposomes via the dehydration-rehydration method. Particle size and polydispersity index (PDI) were measured via dynamic light scattering (DLS), and zeta potential with laser Doppler electrophoresis. Peptide loading was fluorescently determined and the immunogenicity of the formulated peptides was assessed in co-cultures of dendritic cells (DCs) and CD8+ T-cells in vitro. RESULTS: All SLPs were loaded in cationic liposomes by using three different loading method variants, depending on the SLP characteristics. The fifteen liposomal formulations had a comparable size (< 200 nm), PDI (< 0.3) and zeta potential (22-30 mV). Cationic liposomes efficiently delivered the SLPs to DCs that subsequently activated SIINFEKL-specific CD8+ T-cells, indicating improved immunological activity of the SLPs. CONCLUSION: Cationic liposomes can accommodate a wide range of different SLPs and are therefore a potential delivery platform for personalized cancer vaccines.

Efficient Eradication of Established Tumors in Mice with Cationic Liposome-Based Synthetic Long-Peptide Vaccines.

Therapeutic vaccination with synthetic long peptides (SLP) can be clinically effective against HPV-induced premalignant lesions; however, their efficiency in established malignant lesions leaves room for improvement. Here, we report the high therapeutic potency of cationic liposomes loaded with well-defined tumor-specific SLPs and a TLR3 ligand as adjuvant. The cationic particles, with an average size of 160 nm, could strongly activate functional, antigen-specific CD8+ and CD4+ T cells and induced in vivo cytotoxicity against target cells after intradermal vaccination. At a low dose (1 nmol) of SLP, our liposomal formulations significantly controlled tumor outgrowth in two independent models (melanoma and HPV-induced tumors) and even cured 75%-100% of mice of their large established tumors. Cured mice were fully protected from a second challenge with an otherwise lethal dose of tumor cells, indicating the potential of liposomal SLP in the formulation of powerful vaccines for cancer immunotherapy. Cancer Immunol Res; 5(3); 222-33. ©2017 AACR.

Synthetic long peptide-based vaccine formulations for induction of cell mediated immunity: A comparative study of cationic liposomes and PLGA nanoparticles.

Nanoparticulate formulations for synthetic long peptide (SLP)-cancer vaccines as alternative to clinically used Montanide ISA 51- and squalene-based emulsions are investigated in this study. SLPs were loaded into TLR ligand-adjuvanted cationic liposomes and PLGA nanoparticles (NPs) to potentially induce cell-mediated immune responses. The liposomal and PLGA NP formulations were successfully loaded with up to four different compounds and were able to enhance antigen uptake by dendritic cells (DCs) and subsequent activation of T cells in vitro. Subcutaneous vaccination of mice with the different formulations showed that the SLP-loaded cationic liposomes were the most efficient for the induction of functional antigen-T cells in vivo, followed by PLGA NPs which were as potent as or even more than the Montanide and squalene emulsions. Moreover, after transfer of antigen-specific target cells in immunized mice, liposomes induced the highest in vivo killing capacity. These findings, considering also the inadequate safety profile of the currently clinically used adjuvant Montanide ISA-51, make these two particulate, biodegradable delivery systems promising candidates as delivery platforms for SLP-based immunotherapy of cancer.

Cationic liposomes loaded with a synthetic long peptide and poly(I:C): a defined adjuvanted vaccine for induction of antigen-specific T cell cytotoxicity.

For effective cancer immunotherapy by vaccination, co-delivery of tumour antigens and adjuvants to dendritic cells and subsequent activation of antigen-specific cytotoxic T cells (CTLs) is crucial. In this study, a synthetic long peptide (SLP) harbouring the model CTL epitope SIINFEKL was encapsulated with the TLR3 ligand poly(inosinic-polycytidylic acid) (poly(I:C)) in cationic liposomes consisting of DOTAP and DOPC. The obtained particles were down-sized to about 140 nm (measured by dynamic light scattering) and had a positive zeta-potential of about 26 mV (according to laser Doppler electrophoresis). SLP loading efficiency was about 40% as determined by HPLC. Poly(I:C) loading efficiency was about 50%, as assessed from the fluorescence intensity of fluorescently labelled poly(I:C). Immunogenicity of the liposomal SLP vaccine was evaluated in vitro by its capacity to activate dendritic cells (DCs) and present the processed SLP to SIINFEKL-specific T cells. The effectiveness of the vaccine to activate CD8(+) T cells was analysed in vivo after intradermal and subcutaneous immunisation in mice, by measuring antigen-specific T cells in blood and spleens and assessing their functionality by cytokine production and in vivo cytotoxicity. The liposomal formulation efficiently delivered the SLP to DCs in vitro and induced a functional CD8(+) T cell immune response in vivo to the CTL epitope present in the SLP. The SLP-specific CD8(+) T cell frequency induced by the poly(I:C)-adjuvanted liposomal SLP formulation showed an at least 25 fold increase over the T cell frequency induced by the poly(I:C)-adjuvanted soluble SLP. In conclusion, cationic liposomes loaded with SLP and poly(I:C) have potential as a powerful therapeutic cancer vaccine formulation.

Parameter optimization toward optimal microneedle-based dermal vaccination.

Microneedle-based vaccination has several advantages over vaccination by using conventional hypodermic needles. Microneedles are used to deliver a drug into the skin in a minimally-invasive and potentially pain free manner. Besides, the skin is a potent immune organ that is highly suitable for vaccination. However, there are several factors that influence the penetration ability of the skin by microneedles and the immune responses upon microneedle-based immunization. In this study we assessed several different microneedle arrays for their ability to penetrate ex vivo human skin by using trypan blue and (fluorescently or radioactively labeled) ovalbumin. Next, these different microneedles and several factors, including the dose of ovalbumin, the effect of using an impact-insertion applicator, skin location of microneedle application, and the area of microneedle application, were tested in vivo in mice. The penetration ability and the dose of ovalbumin that is delivered into the skin were shown to be dependent on the use of an applicator and on the microneedle geometry and size of the array. Besides microneedle penetration, the above described factors influenced the immune responses upon microneedle-based vaccination in vivo. It was shown that the ovalbumin-specific antibody responses upon microneedle-based vaccination could be increased up to 12-fold when an impact-insertion applicator was used, up to 8-fold when microneedles were applied over a larger surface area, and up to 36-fold dependent on the location of microneedle application. Therefore, these influencing factors should be considered to optimize microneedle-based dermal immunization technologies.

Ovalbumin-coated pH-sensitive microneedle arrays effectively induce ovalbumin-specific antibody and T-cell responses in mice.

The aim of this work was to study the applicability of antigen-coated pH-sensitive microneedle arrays for effective vaccination strategies. Therefore, a model antigen (ovalbumin) was coated onto pH-sensitive (pyridine-modified) microneedle arrays to test pH-triggered antigen release by applying the coated arrays onto ex vivo human skin, and by conducting a dermal immunization study in mice. The release of antigen into ex vivo human skin from the coated microneedles was determined by using radioactively labeled ovalbumin. To investigate the induction of antigen-specific IgG, and CD4(+) and CD8(+) T-cell responses, BALB/c mice were immunized with antigen-coated pH-sensitive microneedles by the 'coat and poke' approach. These responses were compared to responses induced by the 'poke and patch' approach, and subcutaneous and intradermal vaccination with classic hypodermic needles. The pH-sensitive microneedle arrays were efficiently coated with ovalbumin (95% coating efficiency) and upon application of six microneedle arrays 4.27 of 7 µg ovalbumin was delivered into the skin, showing a release efficiency of 70%. In contrast, the 'poke and patch' approach led to a delivery of only 6.91 of 100 µg ovalbumin (7% delivery efficiency). Immunization by means of ovalbumin-coated microneedles resulted in robust CD4(+) and CD8(+) T-cell responses comparable to those obtained after subcutaneous or intradermal immunization with conventional needles. Moreover, it effectively induced IgG responses; however, it required prime-boost immunizations before antibodies were produced. In conclusion, antigen delivery into ex vivo human skin by antigen-coated pH-sensitive microneedle arrays is more efficient than the 'poke-and-patch' approach and in vivo vaccination studies show the applicability of pH-sensitive microneedles for the induction of both T cell and B cell responses.

Macrophages inhibit human osteosarcoma cell growth after activation with the bacterial cell wall derivative liposomal muramyl tripeptide in combination with interferon-γ.

BACKGROUND: In osteosarcoma, the presence of tumor-infiltrating macrophages positively correlates with patient survival in contrast to the negative effect of tumor-associated macrophages in patients with other tumors. Liposome-encapsulated muramyl tripeptide (L-MTP-PE) has been introduced in the treatment of osteosarcoma patients, which may enhance the potential anti-tumor activity of macrophages. Direct anti-tumor activity of human macrophages against human osteosarcoma cells has not been described so far. Hence, we assessed osteosarcoma cell growth after co-culture with human macrophages. METHODS: Monocyte-derived M1-like and M2-like macrophages were polarized with LPS + IFN-γ, L-MTP-PE +/- IFN-γ or IL-10 and incubated with osteosarcoma cells. Two days later, viable tumor cell numbers were analyzed. Antibody-dependent effects were investigated using the therapeutic anti-EGFR antibody cetuximab. RESULTS: M1-like macrophages inhibited osteosarcoma cell growth when activated with LPS + IFN-γ. Likewise, stimulation of M1-like macrophages with liposomal muramyl tripeptide (L-MTP-PE) inhibited tumor growth, but only when combined with IFN-γ. Addition of the tumor-reactive anti-EGFR antibody cetuximab did not further improve the anti-tumor activity of activated M1-like macrophages. The inhibition was mediated by supernatants of activated M1-like macrophages, containing TNF-α and IL-1ß. However, specific blockage of these cytokines, nitric oxide or reactive oxygen species did not inhibit the anti-tumor effect, suggesting the involvement of other soluble factors released upon macrophage activation. While LPS + IFN-γ-activated M2-like macrophages had low anti-tumor activity, IL-10-polarized M2-like macrophages were able to reduce osteosarcoma cell growth in the presence of the anti-EGFR cetuximab involving antibody-dependent tumor cell phagocytosis. CONCLUSION: This study demonstrates that human macrophages can be induced to exert direct anti-tumor activity against osteosarcoma cells. Our observation that the induction of macrophage anti-tumor activity by L-MTP-PE required IFN-γ may be of relevance for the optimization of L-MTP-PE therapy in osteosarcoma patients.

Novel hollow microneedle technology for depth-controlled microinjection-mediated dermal vaccination: a study with polio vaccine in rats.

PURPOSE: The aim of the study was to develop a cheap and fast method to produce hollow microneedles and an applicator for injecting vaccines into the skin at a pre-defined depth and test the applicability of the system for dermal polio vaccination. METHODS: Hollow microneedles were produced by hydrofluoric acid etching of fused silica capillaries. An electromagnetic applicator was developed to control the insertion speed (1-3 m/s), depth (0-1,000 µm), and angle (10°-90°). Hollow microneedles with an inner diameter of 20 µm were evaluated in ex vivo human skin and subsequently used to immunize rats with inactivated poliovirus vaccine (IPV) by an intradermal microinjection of 9 µL at a depth of 300 µm and an insertion speed of 1 m/s. Rat sera were tested for IPV-specific IgG and virus-neutralizing antibodies. RESULTS: Microneedles produced from fused silica capillaries were successfully inserted into the skin to a chosen depth, without clogging or breakage of the needles. Intradermal microinjection of IPV induced immune responses comparable to those elicited by conventional intramuscular immunization. CONCLUSIONS: We successfully developed a hollow microneedle technology for dermal vaccination that enables fundamental research on factors, such as insertion depth and volume, and insertion angle, on the immune response.