Inflammasome-Dependent Induction of Adaptive NK Cell Memory.

Monobenzone is a pro-hapten that is exclusively metabolized by melanocytes, thereby haptenizing melanocyte-specific antigens, which results in cytotoxic autoimmunity specifically against pigmented cells. Studying monobenzone in a setting of contact hypersensitivity (CHS), we observed that monobenzone induced a long-lasting, melanocyte-specific immune response that was dependent on NK cells, yet fully intact in the absence of T- and B cells. Consistent with the concept of "memory NK cells," monobenzone-induced NK cells resided in the liver and transfer of these cells conferred melanocyte-specific immunity to naive animals. Monobenzone-exposed skin displayed macrophage infiltration and cutaneous lymph nodes showed an inflammasome-dependent influx of macrophages with a tissue-resident phenotype, coinciding with local NK cell activation. Indeed, macrophage depletion or the absence of the NLRP3 inflammasome, the adaptor protein ASC or interleukin-18 (IL-18) abolished monobenzone CHS, thereby establishing a non-redundant role for the NLRP3 inflammasome as a critical proinflammatory checkpoint in the induction of hapten-dependent memory NK cells.

Enhanced HPV16 E6/E7+ tumor eradication via induction of tumor-specific T cells by therapeutic vaccination with virosomes presenting synthetic long peptides.

Therapeutic cancer vaccines trigger CD4 + and CD8 + T cell responses capable of established tumor eradication. Current platforms include DNA, mRNA and synthetic long peptide (SLP) vaccines, all aiming at robust T cell responses. SLPs linked to the Amplivant® adjuvant (Amplivant-SLP) have shown effective delivery to dendritic cells, resulting in improved immunogenicity in mice. We have now tested virosomes as a delivery vehicle for SLPs. Virosomes are nanoparticles made from influenza virus membranes and have been used as vaccines for a variety of antigens. Amplivant-SLP virosomes induced the expansion of more antigen-specific CD8 + T memory cells in ex vivo experiments with human PBMCs than Amplivant-SLP conjugates alone. The immune response could be further improved by including the adjuvants QS-21 and 3D-PHAD in the virosomal membrane. In these experiments, the SLPs were anchored in the membrane through the hydrophobic Amplivant adjuvant. In a therapeutic mouse model of HPV16 E6/E7+ cancer, mice were vaccinated with virosomes loaded with either Amplivant-conjugated SLPs or lipid-coupled SLPs. Vaccination with both types of virosomes significantly improved the control of tumor outgrowth, leading to elimination of the tumors in about half the animals for the best combinations of adjuvants and to their survival beyond 100 days.

Antigen processing by nardilysin and thimet oligopeptidase generates cytotoxic T cell epitopes.

Cytotoxic T lymphocytes (CTLs) recognize peptides presented by HLA class I molecules on the cell surface. The C terminus of these CTL epitopes is considered to be produced by the proteasome. Here we demonstrate that the cytosolic endopeptidases nardilysin and thimet oligopeptidase (TOP) complemented proteasome activity. Nardilysin and TOP were required, either together or alone, for the generation of a tumor-specific CTL epitope from PRAME, an immunodominant CTL epitope from Epstein-Barr virus protein EBNA3C, and a clinically important epitope from the melanoma protein MART-1. TOP functioned as C-terminal trimming peptidase in antigen processing, and nardilysin contributed to both the C-terminal and N-terminal generation of CTL epitopes. By broadening the antigenic peptide repertoire, nardilysin and TOP strengthen the immune defense against intracellular pathogens and cancer.

IFN-γ-induced PD-L1 expression on human melanocytes is impaired in vitiligo.

Mounting evidence shows that the PD-1/PD-L1 axis is involved in tumor immune evasion. This is demonstrated by anti-PD-1 antibodies that can reverse tumor-associated PD-L1 to functionally suppress anti-tumor T-cell responses. Since type I and II interferons are key regulators of PD-L1 expression in melanoma cells and IFN-γ-producing CD8+ T cells and IFN-α-producing dendritic cells are abundant in vitiligo skin, we aimed to study the role of PD-1/PD-L1 signalling in melanocyte destruction in vitiligo. Moreover, impaired PD-1/PD-L1 function is observed in a variety of autoimmune diseases. It is, therefore, hypothesized that manipulating PD-1/PD-L1 signalling might have therapeutic potential in vitiligo. The PD-1+ T cells were abundantly present in situ in perilesional vitiligo skin, but expression of PD-L1 was limited and confined exclusively to dermal T cells. More specifically, neither melanocytes nor other epidermal skin cells expressed PD-L1. Exposure to IFN-γ, but also type I interferons, increased PD-L1 expression in primary melanocytes and fibroblasts, derived from healthy donors. Primary human keratinocytes only showed increased PD-L1 expression upon stimulation with IFN-γ. More interestingly, melanocytes derived from non-lesional vitiligo skin showed no PD-L1 upregulation upon IFN-γ exposure, while other skin cells displayed significant PD-L1 expression after exposure. In a vitiligo skin explant model, incubation of non-lesional vitiligo skin with activated (IFN-γ-producing) T cells from vitiligo lesions was previously described to induce melanocyte apoptosis. Although PD-L1 expression was induced in epidermal cells in these explants, this induction was completely absent in melanocytes. The lack of PD-L1 upregulation by melanocytes in the presence of IFN-γ-producing T cells shows that melanocytes lack protection against T-cell attack during vitiligo pathogenesis. Manipulating PD-1/PD-L1 signalling may, therefore, be a therapeutic option for vitiligo patients.

Formation of Immune Complexes with a Tetanus-Derived B Cell Epitope Boosts Human T Cell Responses to Covalently Linked Peptides in an Ex Vivo Blood Loop System.

Enhancing T cell responses against both viral and tumor Ags requires efficient costimulation and directed delivery of peptide Ags into APCs. Long peptide vaccines are considered favorable vaccine moieties from a clinical perspective, as they can harbor more than one immunogenic epitope enabling treatment of a broader target population. In addition, longer peptides are not extracellularly loaded on MHC class I; rather, they require intracellular processing and will thereby be presented to T cells mainly by professional APCs, thereby avoiding the risk of tolerance induction. The drawback of peptide vaccines regardless of peptide length is that naked peptides are not actively targeted to and taken up by APCs, and the standard nonconjugated adjuvant-peptide mixtures do not ensure cotargeting of the two to the same APC. We have identified a tetanus toxin-derived B cell epitope that can mediate the formation of immune complexes in the presence of circulating Abs. In this study, we show that these immune complexes improve both Ag uptake by APCs (blood monocytes and CD1c+ dendritic cells) and consequently improve CD8+ T cell recall responses in a human ex vivo blood loop system. The uptake of the peptide conjugate by blood monocytes is dependent on Abs and the complement component C1q. We envision that this strategy can be used to facilitate active uptake of Ags into APCs to improve T cell responses against pathogens or cancer.

Checkpoint blockade cancer immunotherapy targets tumour-specific mutant antigens.

The immune system influences the fate of developing cancers by not only functioning as a tumour promoter that facilitates cellular transformation, promotes tumour growth and sculpts tumour cell immunogenicity, but also as an extrinsic tumour suppressor that either destroys developing tumours or restrains their expansion. Yet, clinically apparent cancers still arise in immunocompetent individuals in part as a consequence of cancer-induced immunosuppression. In many individuals, immunosuppression is mediated by cytotoxic T-lymphocyte associated antigen-4 (CTLA-4) and programmed death-1 (PD-1), two immunomodulatory receptors expressed on T cells. Monoclonal-antibody-based therapies targeting CTLA-4 and/or PD-1 (checkpoint blockade) have yielded significant clinical benefits-including durable responses--to patients with different malignancies. However, little is known about the identity of the tumour antigens that function as the targets of T cells activated by checkpoint blockade immunotherapy and whether these antigens can be used to generate vaccines that are highly tumour-specific. Here we use genomics and bioinformatics approaches to identify tumour-specific mutant proteins as a major class of T-cell rejection antigens following anti-PD-1 and/or anti-CTLA-4 therapy of mice bearing progressively growing sarcomas, and we show that therapeutic synthetic long-peptide vaccines incorporating these mutant epitopes induce tumour rejection comparably to checkpoint blockade immunotherapy. Although mutant tumour-antigen-specific T cells are present in progressively growing tumours, they are reactivated following treatment with anti-PD-1 and/or anti-CTLA-4 and display some overlapping but mostly treatment-specific transcriptional profiles, rendering them capable of mediating tumour rejection. These results reveal that tumour-specific mutant antigens are not only important targets of checkpoint blockade therapy, but they can also be used to develop personalized cancer-specific vaccines and to probe the mechanistic underpinnings of different checkpoint blockade treatments.

HBV-Derived Synthetic Long Peptide Can Boost CD4+ and CD8+ T-Cell Responses in Chronic HBV Patients Ex Vivo.

Background: Vaccination with synthetic long peptides (SLP) is a promising new treatment strategy for chronic hepatitis B virus (CHB). SLP can induce broad T-cell responses for all HLA types. Here we investigated the ability of a prototype HBV-core (HBc)-sequence-derived SLP to boost HBV-specific T cells in CHB patients ex vivo. Methods: HBc-SLP was used to assess cross-presentation by monocyte-derived dendritic cells (moDC) and BDCA1+ blood myeloid DC (mDC) to engineered HBV-specific CD8+ T cells. Autologous SLP-loaded and toll-like receptor (TLR)-stimulated DC were used to activate patient HBc-specific CD8+ and CD4+ T cells. Results: HBV-SLP was cross-presented by moDC, which was further enhanced by adjuvants. Patient-derived SLP-loaded moDC significantly increased autologous HBcAg18-27-specific CD8+ T cells and CD4+ T cells ex vivo. HBV-specific T cells were functional as they synthesized tumor necrosis factor-alpha and interferon-gamma. In 6/7 of patients blockade of PD-L1 further increased SLP effects. Also, importantly, patient-derived BDCA1+ mDC cross-presented and activated autologous T-cell responses ex vivo. Conclusions: As a proof of concept, we showed a prototype HBc-SLP can boost T-cell responses in patients ex vivo. These results pave the way for the development of a therapeutic SLP-based vaccine to induce effective HBV-specific adaptive immune responses in CHB patients.

Cancer immunotherapy by dendritic cells.

Cancerous lesions promote tumor growth, motility, invasion, and angiogenesis via oncogene-driven immunosuppressive leukocyte infiltrates, mainly myeloid-derived suppressor cells, tumor-associated macrophages, and immature dendritic cells (DCs). In addition, many tumors express or induce immunosuppressive cytokines such as TGF-beta and IL-10. As a result, tumor-antigen crosspresentation by DCs induces T cell anergy or deletion and regulatory T cells instead of antitumor immunity. Tumoricidal effector cells can be generated after vigorous DC activation by Toll-like receptor ligands or CD40 agonists. However, no single immunotherapeutic modality is effective in established cancer. Rather, chemotherapies, causing DC activation, enhanced crosspresentation, lymphodepletion, and reduction of immunosuppressive leukocytes, act synergistically with vaccines or adoptive T cell transfer. Here, I discuss the considerations for generating promising therapeutic antitumor vaccines that use DCs.

Prospects of combinatorial synthetic peptide vaccine-based immunotherapy against cancer.

The insight that the immune system is involved in tumor resistance is gaining momentum and this has led to the development of immunotherapeutic strategies aiming at enhancement of immune-mediated tumor destruction. Although some of these strategies have moderate clinical benefit, most stand-alone therapies fail to significantly affect progressive disease and survival or do so only in a minority of patients. Research on the mechanisms underlying the generation of immune responses against tumors and the immune evasion by tumors has emphasized that various mechanisms simultaneously prevent effective immunity against cancer including inefficient presentation of tumor antigens by dendritic cells and induction of negative immune regulation by regulatory T-cells (Tregs) and myeloid derived suppressor cells (MDSCs). Thus the design of therapies that simultaneously improve effective tumor immunity and counteract immune evasion by tumors seems most desirable for clinical efficacy. As it is unlikely that a single immunotherapeutic strategy addresses all necessary requirements, combinatorial strategies that act synergistically need to be developed. Here we discuss the current knowledge and prospects of treatment with synthetic peptide vaccines that stimulate tumor-specific T-cell responses combined with adjuvants, immune modulating antibodies, cytokines and chemotherapy. We conclude that combinatorial approaches have the best potency to accomplish the most significant tumor destruction but further research is required to optimize such approaches.

IgG-mediated anaphylaxis to a synthetic long peptide vaccine containing a B cell epitope can be avoided by slow-release formulation.

Synthetic long peptides (SLP) are a promising vaccine modality to induce therapeutic T cell responses in patients with chronic infections and tumors. We studied different vaccine formulations in mice using SLP derived from carcinoembryonic Ag. We discovered that one of the SLP contains a linear Ab epitope in combination with a CD4 epitope. Repeated vaccination with this carcinoembryonic Ag SLP in mice shows improved T cell responses and simultaneously induced high titers of peptide-specific Abs. These Abs resulted in unexpected anaphylaxis after a third or subsequent vaccinations with the SLP when formulated in saline. Administration of low SLP doses in the slow-release vehicle IFA prevented the anaphylaxis after repeated vaccination. This study underscores both the immunogenicity of SLP vaccination, for inducing T cell as well as B cell responses, and the necessity of safe administration routes.

Synthetic long peptide booster immunization in rhesus macaques primed with replication-competent NYVAC-C-KC induces a balanced CD4/CD8 T-cell and antibody response against the conserved regions of HIV-1.

The Thai trial (RV144) indicates that a prime-boost vaccine combination that induces both T-cell and antibody responses may be desirable for an effective HIV vaccine. We have previously shown that immunization with synthetic long peptides (SLP), covering the conserved parts of SIV, induced strong CD4 T-cell and antibody responses, but only modest CD8 T-cell responses. To generate a more balanced CD4/CD8 T-cell and antibody response, this study evaluated a pox-vector prime/SLP boost strategy in rhesus macaques. Priming with a replication-competent NYVAC, encoding HIV-1 clade C gag, pol and nef, induced modest IFNγ T-cell immune responses, predominantly directed against HIV-1 Gag. Booster immunization with SLP, covering the conserved parts of HIV-1 Gag, Pol and Env, resulted in a more than 10-fold increase in IFNγ ELISpot responses in four of six animals, which were predominantly HIV-1 Pol-specific. The animals showed a balanced polyfunctional CD4 and CD8 T-cell response and high Ab titres.

Human papillomavirus (HPV) upregulates the cellular deubiquitinase UCHL1 to suppress the keratinocyte's innate immune response.

Persistent infection of basal keratinocytes with high-risk human papillomavirus (hrHPV) may cause cancer. Keratinocytes are equipped with different pattern recognition receptors (PRRs) but hrHPV has developed ways to dampen their signals resulting in minimal inflammation and evasion of host immunity for sustained periods of time. To understand the mechanisms underlying hrHPV's capacity to evade immunity, we studied PRR signaling in non, newly, and persistently hrHPV-infected keratinocytes. We found that active infection with hrHPV hampered the relay of signals downstream of the PRRs to the nucleus, thereby affecting the production of type-I interferon and pro-inflammatory cytokines and chemokines. This suppression was shown to depend on hrHPV-induced expression of the cellular protein ubiquitin carboxyl-terminal hydrolase L1 (UCHL1) in keratinocytes. UCHL1 accomplished this by inhibiting tumor necrosis factor receptor-associated factor 3 (TRAF3) K63 poly-ubiquitination which lead to lower levels of TRAF3 bound to TANK-binding kinase 1 and a reduced phosphorylation of interferon regulatory factor 3. Furthermore, UCHL1 mediated the degradation of the NF-kappa-B essential modulator with as result the suppression of p65 phosphorylation and canonical NF-κB signaling. We conclude that hrHPV exploits the cellular protein UCHL1 to evade host innate immunity by suppressing PRR-induced keratinocyte-mediated production of interferons, cytokines and chemokines, which normally results in the attraction and activation of an adaptive immune response. This identifies UCHL1 as a negative regulator of PRR-induced immune responses and consequently its virus-increased expression as a strategy for hrHPV to persist.

Dendritic cells process synthetic long peptides better than whole protein, improving antigen presentation and T-cell activation.

The efficiency of antigen (Ag) processing by dendritic cells (DCs) is vital for the strength of the ensuing T-cell responses. Previously, we and others have shown that in comparison to protein vaccines, vaccination with synthetic long peptides (SLPs) has shown more promising (pre-)clinical results. Here, we studied the unknown mechanisms underlying the observed vaccine efficacy of SLPs. We report an in vitro processing analysis of SLPs for MHC class I and class II presentation by murine DCs and human monocyte-derived DCs. Compared to protein, SLPs were rapidly and much more efficiently processed by DCs, resulting in an increased presentation to CD4⁺ and CD8⁺ T cells. The mechanism of access to MHC class I loading appeared to differ between the two forms of Ag. Whereas whole soluble protein Ag ended up largely in endolysosomes, SLPs were detected very rapidly outside the endolysosomes after internalization by DCs, followed by proteasome- and transporter associated with Ag processing-dependent MHC class I presentation. Compared to the slower processing route taken by whole protein Ags, our results indicate that the efficient internalization of SLPs, accomplished by DCs but not by B or T cells and characterized by a different and faster intracellular routing, leads to enhanced CD8⁺ T-cell activation.

The long-term immune response after HPV16 peptide vaccination in women with low-grade pre-malignant disorders of the uterine cervix: a placebo-controlled phase II study.

The capacity of a low-dose HPV16 synthetic long-peptide vaccine (HPV16-SLP) to induce an HPV16-specific T-cell response as well as to establish long-term immunologic memory in patients with low-grade abnormalities of the cervix was determined in a placebo-controlled, double-blinded phase II study. In addition, the effect of a booster vaccination after 1 year was evaluated. Patients received either the HPV16-SLP or a placebo at the start of the study. After 1 year, the vaccinated patients were again randomized to receive the HPV16-SLP or a placebo. Patients were followed for 2 years. HPV16-specific T-cell responses were determined in pre- and post-vaccination blood samples by ELISPOT, proliferation assay and cytokine assays. We show that the HPV16-specific T-cell responses detected after vaccination are clearly due to vaccination and that reactivity was maintained for at least 2 years. Interestingly, a booster vaccination after 1 year especially augmented the HPV16-specific Th2 response. Furthermore, pre-existing immunity to HPV16 was associated with a stronger response to vaccination and with more side effects, reflected by flu-like symptoms. We conclude that two low-dose injections of HPV16-SLP can induce a strong and stable HPV16-specific T-cell response that lasts for at least 1 year. If booster vaccination is required, then polarizing adjuvant should be added to maintain the Th1 focus of the vaccine-induced T-cell response.

CD27-CD70 costimulation controls T cell immunity during acute and persistent cytomegalovirus infection.

Cytomegaloviruses (CMVs) establish lifelong infections that are controlled in part by CD4(+) and CD8(+) T cells. To promote persistence, CMVs utilize multiple strategies to evade host immunity, including modulation of costimulatory molecules on infected antigen-presenting cells. In humans, CMV-specific memory T cells are characterized by the loss of CD27 expression, which suggests a critical role of the costimulatory receptor-ligand pair CD27-CD70 for the development of CMV-specific T cell immunity. In this study, the in vivo role of CD27-CD70 costimulation during mouse CMV infection was examined. During the acute phase of infection, the magnitudes of CMV-specific CD4(+) and CD8(+) T cell responses were decreased in mice with abrogated CD27-CD70 costimulation. Moreover, the accumulation of inflationary memory T cells during the persistent phase of infection and the ability to undergo secondary expansion required CD27-CD70 interactions. The downmodulation of CD27 expression, however, which occurs gradually and exclusively on inflationary memory T cells, is ligand independent. Furthermore, the IL-2 production in both noninflationary and inflationary CMV-specific T cells was dependent on CD27-CD70 costimulation. Collectively, these results highlight the importance of the CD27-CD70 costimulation pathway for the development of CMV-specific T cell immunity during acute and persistent infection.

Immunotherapy for persistent viral infections and associated disease.

Persistent viral infections reflect a failure of the host's immune system to control infection, and in many cases, they are associated with the development of malignancies. So far, vaccines designed to boost viral immunity during chronic infection have not been successful. Infections with high-risk human papilloma viruses (e.g. HPV16) are acquired by a large segment of the population and persist in 5-10% of infected individuals, which causes the development of high-grade pre-malignant lesions. Recently we succeeded in causing regression of HPV16-induced disease in ∼50% of chronically infected patients by a novel therapeutic vaccine. Here, we summarize the parallels in immunity against HPV and other chronic viruses and discuss the general implications of our findings for the immunotherapy of chronic infections.

Vaccine-induced effector-memory CD8+ T cell responses predict therapeutic efficacy against tumors.

CD8(+) T cells have the potential to attack and eradicate cancer cells. The efficacy of therapeutic vaccines against cancer, however, lacks defined immune correlates of tumor eradication after (therapeutic) vaccination based on features of Ag-specific T cell responses. In this study, we examined CD8(+) T cell responses elicited by various peptide and TLR agonist-based vaccine formulations in nontumor settings and show that the formation of CD62L(-)KLRG1(+) effector-memory CD8(+) T cells producing the effector cytokines IFN-γ and TNF predicts the degree of therapeutic efficacy of these vaccines against established s.c. tumors. Thus, characteristics of vaccine-induced CD8(+) T cell responses instill a predictive determinant for the efficacy of vaccines during tumor therapy.