Antigen-specific Immunotherapy Research Articles

Carfilzomib alters the HLA-presented peptidome of myeloma cells and impairs presentation of peptides with aromatic C-termini.

Recent studies suggest that multiple myeloma is an immunogenic disease, which might be effectively targeted by antigen-specific T-cell immunotherapy. As standard of care in myeloma includes proteasome inhibitor therapy, it is of great importance to characterize the effects of this treatment on HLA-restricted antigen presentation and implement only robustly presented targets for immunotherapeutic intervention. Here, we present a study that longitudinally and semi-quantitatively maps the effects of the proteasome inhibitor carfilzomib on HLA-restricted antigen presentation. The relative presentation levels of 4780 different HLA ligands were quantified in an in vitro model employing carfilzomib treatment of MM.1S and U266 myeloma cells, which revealed significant modulation of a substantial fraction of the HLA-presented peptidome. Strikingly, we detected selective down-modulation of HLA ligands with aromatic C-terminal anchor amino acids. This particularly manifested as a marked reduction in the presentation of HLA ligands through the HLA allotypes A*23:01 and A*24:02 on MM.1S cells. These findings implicate that carfilzomib mediates a direct, peptide motif-specific inhibitory effect on HLA ligand processing and presentation. As a substantial proportion of HLA allotypes present peptides with aromatic C-termini, our results may have broad implications for the implementation of antigen-specific treatment approaches in patients undergoing carfilzomib treatment.

Autoimmunity: Nanoparticles engineered for antigen-specific immunotherapy.

Autoimmunity: Nanoparticles engineered for antigen-specific immunotherapy.

Sa1395 Nexvax2®, a Peptide-Based Antigen-Specific Immunotherapy, Administered Intra-Dermally Three-Times Over 15-Days attenuates Responsiveness to Immuno-Dominant Gluten Peptides in HLA-DQ2.5+ People With Celiac Disease (CeD)

Sa1395 Nexvax2®, a Peptide-Based Antigen-Specific Immunotherapy, Administered Intra-Dermally Three-Times Over 15-Days attenuates Responsiveness to Immuno-Dominant Gluten Peptides in HLA-DQ2.5+ People With Celiac Disease (CeD)

Autoimmunity: Nanoparticles engineered for antigen-specific immunotherapy.

Autoimmunity: Nanoparticles engineered for antigen-specific immunotherapy.

Remarkable Success in Treating Autoimmune Diseases in Mice

The Holy Grail of treating autoimmune diseases has been suppressing an immune attack on a target organ without compromising systemic immunity. An international research team reports having achieved such antigen-specific immunotherapy in mice. The team used mouse models of three autoimmune diseases: type 1 diabetes, experimental autoimmune encephalomyelitis (EAE; a …

Identification of Novel HLA-A*24:02-Restricted Epitope Derived from a Homeobox Protein Expressed in Hematological Malignancies.

The homeobox protein, PEPP2 (RHOXF2), has been suggested as a cancer/testis (CT) antigen based on its expression pattern. However, the peptide epitope of PEPP2 that is recognized by cytotoxic T cells (CTLs) is unknown. In this study, we revealed that PEPP2 gene was highly expressed in myeloid leukemia cells and some other hematological malignancies. This gene was also expressed in leukemic stem-like cells. We next identified the first reported epitope peptide (PEPP2271-279). The CTLs induced by PEPP2271-279 recognized PEPP2-positive target cells in an HLA-A*24:02-restricted manner. We also found that a demethylating agent, 5-aza-2’-deoxycytidine, could enhance PEPP2 expression in leukemia cells but not in blood mononuclear cells from healthy donors. The cytotoxic activity of anti-PEPP2 CTL against leukemic cells treated with 5-aza-2’-deoxycytidine was higher than that directed against untreated cells. These results suggest a clinical rationale that combined treatment with this novel antigen-specific immunotherapy together with demethylating agents might be effective in therapy-resistant myeloid leukemia patients.

Molecular Dynamics of Multivalent Soluble Antigen Arrays Support a Two-Signal Co-delivery Mechanism in the Treatment of Experimental Autoimmune Encephalomyelitis.

Many current therapies for autoimmune diseases such as multiple sclerosis (MS) result in global immunosuppression, rendering insufficient efficacy with increased risk of adverse side effects. Multivalent soluble antigen arrays, nanomaterials presenting both autoantigen and secondary inhibitory signals on a flexible polymer backbone, are hypothesized to shift the immune response toward selective autoantigenic tolerance to repress autoimmune disease. Two-signal co-delivery of both autoantigen and secondary signal were deemed necessary for therapeutic efficacy against experimental autoimmune encephalomyelitis, a murine model of MS. Dynamic light scattering and in silico molecular dynamics simulations complemented these studies to illuminate the role of two-signal co-delivery in determining therapeutic potential. Physicochemical characteristics such as particle size and molecular affinity for intermolecular interactions and chain entanglement likely facilitated cotransport of two signals to produce efficacy. These findings elucidate potential mechanisms whereby soluble antigen arrays enact their therapeutic effect and help to guide the development of future multivalent antigen-specific immunotherapies.

スギ・ヒノキ花粉症に対するスギ抗原特異的皮下免疫療法の有効性の検討

スギ・ヒノキ花粉症に対するスギ抗原特異的皮下免疫療法の有効性の検討

Generation of BiKEs and TriKEs to Improve NK Cell-Mediated Targeting of Tumor Cells.

Cancer immunotherapies have gained significant momentum over the past decade, particularly with the advent of checkpoint inhibitors and CAR T-cells. While the latter personalized targeted immunotherapy has revolutionized the field, a need for off-the-shelf therapies remains. The ability of NK cells to quickly lyse antibody-coated tumors and potently secrete cytokines without prior priming has made NK cells ideal candidates for antigen-specific immunotherapy. NK cells have been targeted to tumors through two main strategies: mono-specific antibodies and bi/tri-specific antibodies. Mono-specific antibodies drive NK cell antibody-dependent cell-mediated cytotoxicity (ADCC) of tumor cells. Bi/tri-specific antibodies drive re-directed lysis of tumor cells through binding of a tumor antigen and direct binding and crosslinking of the CD16 receptor on NK cells, thus bypassing the need for binding of the Fc portion of mono-specific antibodies. This chapter focuses on the generation of bi- and tri-specific killer engagers (BiKEs and TriKEs) meant to target NK cells to tumors. BiKEs and TriKEs are smaller molecules composed of 2-3 variable portions of antibodies with different specificities, and represent a novel and more versatile strategy compared to traditional bi- and tri-specific antibody platforms.

Multivalent nanomaterial arrays leverage antigen specificity to modulate immune response in autoimmune disease

Event Abstract Back to Event Multivalent nanomaterial arrays leverage antigen specificity to modulate immune response in autoimmune disease Brittany L. Hartwell1, Heather Shinogle2, Bradley P. Sullivan3 and Cory Berkland1, 3, 4 1 University of Kansas, Bioengineering, United States 2 University of Kansas, Microscopy and Analytical Imaging Lab, United States 3 University of Kansas, Pharmaceutical Chemistry, United States 4 University of Kansas, Chemical and Petroleum Engineering, United States Introduction: Many current therapies for autoimmune diseases such as multiple sclerosis (MS) act through nonspecific targeting of the immune response, rendering global immunosuppression, poor efficacy, and adverse side effects. To address a pressing need for safer and more effective therapies, multivalent soluble antigen arrays (SAgAs) have been developed to induce antigen-specific tolerance in MS. SAgAs consist of a flexible hyaluronic acid (HA) linear polymer cografted with multiple copies of autoantigen (proteolipid protein peptide, PLP­139-151) and secondary inhibitory signal (ICAM-1 inhibitor peptide, LABL). These peptides are intended to target SAgA molecules to the immunological synapse, interrupting signaling between T cells and antigen presenting cells (APCs) to halt propagation of an autoimmune attack[1]-[4]. Previous in vivo studies showed SAgAs containing both signals were therapeutically effective against EAE (murine model of MS)[5]. We hypothesize SAgAs enact a therapeutic effect due to enhanced, antigen-specific binding with APCs. To investigate this hypothesis, in vitro studies were performed in a model APC system to investigate antigen-specific binding and receptor response; ex vivo studies were performed in murine splenocytes to investigate targeting of EAE immune cells. Methods: 16 kDa HA was labeled with fluorescein isothiocyanate (FITC) to form ‘fHA’, then conjugated with aminooxy-LABL and/or -PLP to achieve a valency of ~10 peptides per HA backbone. Homopolymers (fHAPLP and fHALABL) were grafted with one peptide and a heteropolymer (fSAgAPLP:LABL) was co-grafted with both peptides. Conjugation was determined by RP-HPLC. In vitro binding studies were performed in Raji B cells (APCs) using flow cytometry. Cells were primed and activated with TNF-α and PLP for 24 hours. To observe maximum binding, cells were mixed with labeled arrays immediately before injection on the cytometer and allowed to reach maximum steady state (SS). Specific binding was then determined through competitive dissociation with excess addition of unlabeled HA, PLP, or LABL. Cell binding and receptor clustering were observed in real-time by fluorescence microscopy using the CellASIC ONIX Microfluidics Platform and M04S switching plate. Ex vivo binding studies were performed in splenocytes isolated from the spleens of both healthy and sick SJL/J mice, harvested at peak of disease following in vivo EAE induction. Isolated splenocytes were stained with CD3, CD22, and CD11c antibodies to identify T cells, B cells, and dendritic cells, respectively. Binding was evaluated by flow cytometry and fluorescence microscopy, as detailed above. Results and Discussion: fSAgAPLP:LABL exhibited significantly greater binding in vitro than fHA, fHAPLP, and fHALABL. Specific binding determined by competitive dissociation was driven by PLP. Receptor clustering, observed using real-time fluorescence microscopy, is an important precursor for signaling and activation of an antigen-specific cellular response. fSAgA­­PLP:LABL induced mature receptor clustering in Raji B cells to a greater extent than fHA, fHAPLP, or fHALABL. fSAgAPLP:LABL exhibited greater binding ex vivo than other arrays and greater binding in EAE splenocytes than healthy splenocytes. Conclusions: SAgAPLP:LABL exhibited enhanced cell binding due to the presence of both PLP and LABL signals, primarily driven by the antigen. SAgAPLP:LABL binding was accompanied by progressive receptor clustering which may contribute to modulation of the immune response. Additionally, SAgAPLP:LABL exhibited greater binding with EAE splenocytes over healthy splenocytes. Leveraging enhanced, antigen-specific binding and receptor modulation, SAgAs offer a promising option for antigen-specific immunotherapy to repress autoimmune disease.

Microneedle delivery of autoantigen for immunotherapy in type 1 diabetes

Antigen specific immunotherapy mediated via the sustained generation of regulatory T cells arguably represents the ideal therapeutic approach to preventing beta cell destruction in type 1 diabetes. However, there is a need to enhance the efficacy of this approach to achieve disease modification in man. Previous studies suggest that prolonged expression of self-antigen in skin in a non-inflammatory context is beneficial for tolerance induction. We therefore sought to develop a dry-coated microneedle (MN) delivery system and combine it with topical steroid to minimise local inflammation and promote prolonged antigen presentation in the skin. Here we show that a combination of surface-modified MNs coated with appropriate solvent systems can deliver therapeutically relevant quantities of peptide to mouse and human skin even with hydrophobic peptides. Compared to conventional “wet” intradermal (ID) administration, “dry” peptide delivered via MNs was retained for longer in the skin and whilst topical hydration of the skin with vehicle or steroid accelerated loss of ID-delivered peptide from the skin, MN delivery of peptide was unaffected. Furthermore, MN delivery resulted in enhanced presentation of antigen to T cells in skin draining lymph nodes (LNs) both 3 and 10days after administration. Repeated administration of islet antigen peptide via MN was effective at reducing antigen-specific T cell proliferation in the pancreatic LN, although topical steroid therapy did not enhance this. Taken together, these data show auto-antigenic peptide delivery into skin using coated MNs results in prolonged retention and enhanced antigen presentation compared to conventional ID delivery and this approach may have potential in individuals identified as being at a high risk of developing type 1 diabetes and other autoimmune diseases.

Investigation of FOXM1 as a Potential New Target for Melanoma.

Recent studies have shown that immunotherapies and molecular targeted therapies are effective for advanced melanoma. Non-antigen-specific immunotherapies such as immunocheckpoint blockades have been shown to be effective in the treatment of advanced melanoma. However, the response rates remain low. To improve their efficacy, they should be combined with antigen-specific immunotherapy. Elevated expression of the transcription factor, Forkhead box M1 (FOXM1), has been reported in various human cancers, and it has been shown to have potential as a target for immunotherapy. The purpose of this study was to investigate the FOXM1 expression in human melanoma samples and cell lines, to evaluate the relationship between the FOXM1 expression and the clinical features of melanoma patients and to investigate the association between the FOXM1 and MAPK and PI3K/AKT pathways in melanoma cell lines. We conducted the quantitative reverse transcription PCR (qRT-PCR) and Western blotting analyses of melanoma cell lines, and investigated melanoma and nevus tissue samples by qRT-PCR and immunohistochemistry. We performed MEK siRNA and PI3K/AKT inhibitor studies and FOXM1 siRNA studies in melanoma cell lines. We found that FOXM1 was expressed in all of the melanoma cell lines, and was expressed in 49% of primary melanomas, 67% of metastatic melanomas and 10% of nevi by performing immunohistochemical staining. Metastatic melanoma samples exhibited significantly higher mRNA levels of FOXM1 (p = 0.004). Primary melanomas thicker than 2 mm were also more likely to express FOXM1. Patients whose primary melanoma expressed FOXM1 had a significantly poorer overall survival compared to patients without FOXM1 expression (p = 0.024). Downregulation of FOXM1 by siRNA significantly inhibited the proliferation of melanoma cells, and blockade of the MAPK and PI3K/AKT pathways decreased the FOXM1 expression in melanoma cell lines. In conclusion, FOXM1 is considered to be a new therapeutic target for melanoma.

Improved Killing of AML Blasts By Dual-Targeting of CD123 and CD33 Via Unitarg a Novel Antibody-Based Modular T Cell Retargeting System

Acute myeloid leukemia (AML) is a hematologic malignancy of the myeloid line with high prevalence in older patients. As complete eradication of metastatic cancer cells is often not achieved by standard therapies, alternative treatment modalities are urgently needed.In recent years, bispecific antibodies (bsAbs) and chimeric antigen receptors (CARs) emerged as promising candidates for an antigen-specific cancer immunotherapy. Both bsAbs and CARs are able to redirect T cells for efficient tumor cell lysis. Nevertheless, the development of a novel TAA specific bsAb or a CAR is a long lasting process. Therefore, we recently introduced a novel antibody-based modular platform (UniTARG) that can be rapidly and easily adapted for redirection of T cells to any TAA in both a bsAb or CAR related manner. The modular UniTARG system distributes the effector arm (the anti-CD3 domain or CAR) and the anti-TAA binding domain to two separate molecules: (I) an exchangeable target module (TM) comprising an anti-TAA binding moiety and a short peptide epitope (E5B9), and (II) a universal effector unit. The effector systems represent either a bsAb with specificity for CD3 and a peptide epitope (E5B9) termed UniMAB or a CAR directed to the E5B9 epitope (UniCAR). Thus, TMs can form a complex with the respective effector system that facilitates the cross-linkage of tumor and T cells similar to conventional bsAbs or CARs. For redirection of T cells to any kind of TAA only the binding moiety of the TM has to be adapted what saves costs and time. To increase tumor specificity and to reduce the risk of tumor escape variants, the modular UniTARG system further offers the possibility to apply simultaneously different monospecific or even bispecific TMs recognizing two TAAs.For proof of concept of a dual targeting using the UniTARG system we selected as TAA on AML blasts the molecules CD33 and CD123. They represent promising target antigens as they are overexpressed on both rapidly proliferating terminal AML blasts and leukemic stem cells which might be responsible for disease relapse after initial chemotherapy. Thus, we generated an anti-CD123 TM and anti-CD33 TM that can be applied within the modular system for single-targeting or that can be combined for dual-targeting of AML blasts. By fusion of the anti-CD123 and anti-CD33 domains via the E5B9 epitope a bispecific TM was further constructed. As revealed by cytotoxicity assays with CD33+ CD123+ AML cell lines, the novel mono- and bispecific TMs can be easily applied to the modular systems to trigger highly potent tumor cell lysis at low E:T ratios and picomolar Ab concentrations. By using the dual-targeting approach we can show that lysis of CD123+ CD33+ AML blasts can be considerably improved in comparison to the mono-specific strategy.Overall, due to the ease and cost-effectiveness of development the UniTARG platform technology represents a promising tool in the field of both bsAbs and CARs with the advantage of simultaneous or consecutive dual or even multispecific targeting. This approach might additionally improve anti-tumor activity by increasing tumor specificity and diminishing off-target effects. DisclosuresCartellieri:Cellex Patient Treatment GmbH: Employment. Ehninger:GEMoaB Monoclonals GmbH: Employment, Patents & Royalties: related to the UniTARG system. Ehninger:GEMoaB Monoclonals GmbH: Equity Ownership, Patents & Royalties: related to the UniTARG system. Bachmann:GEMoaB Monoclonals GmbH: Equity Ownership, Patents & Royalties: related to the UniTARG system.

Interplay Between the Immune System and Colorectal Carcinoma - Towards Tumor-Specific Peptide-Based Vaccination for Any HLA-Type

Introduction: Peptide-basedcancer vaccines targeting tumor-associated antigens comprised of synthetic peptides have shown promising clinical results and have progressed to Phase III clinical testing in several tumor entities, including colorectal carcinoma (CRC). As of yet, these vaccines have typically been limited to HLA-A*02 positive patients, which means that more than half of the patient pool will not be eligible for those treatment strategies. The development of novel strategies for vaccine design, which are applicable to a larger fraction of potential patients, is therefore required. CRC seems to be particularly suitable for antigen-specific immunotherapy as it is among those cancers with highest rates of genetic mutations leading to alterations in protein metabolism. In this study, we aimed to identify HLA class I and II tumor-associated antigens from CRC covering the most frequent HLA-allotypes, in order to develop a peptide warehouse containing HLA-specific peptide panels that can be compiled in a patient-individualized manner, according to the respective patient's HLA-typing.Methods: The HLA presented immunopeptidome of 30 primary CRC samples and matched autologous non-malignant colon tissue was analyzed after HLA-immunoprecipitation by uHPLC tandem mass spectrometry. Identified source proteins and peptides were cross-evaluated with an in-house database of ligandome data derived from different benign tissues to preclude off-tumor presentation of HLA-ligands in order to to prevent any possible induction of autoimmunity by antigen-specific T cells against these ligands. Furthermore, we evaluated pre-existing antigen-specific T cell responses against these novel CRC-associated peptides in a cohort of 25 CRC patients.Results: About 17,000 MHC class I presented peptides could be identified on CRC and were cross-evaluated with our in-house database containing 39,000 MHC class I peptides presented on non-malignant tissue. The ligands identified were derived from 7,500 unique tumor-associated proteins and from 11,500 tumor-exclusive source proteins, respectively. Moreover, about 2,000 different MHC class II presented peptides from more than 1,600 source proteins were identified on CRC tissue. Interestingly, MHC class II peptides could contain shorter amino-acid sequences which - after further proteasomal degradation - might also bind to MHC class I molecules. For clinical applicability, we prioritized the identified ligands according to their frequencies of presentation and detectability on CRC. For representation in the HLA-specific peptide sets, a cutoff expression on at least 25% of allotype-matched tumors was required. This guided the assembly of a peptide vaccine warehouse consisting of 40 highly specific CRC-associated HLA ligands. Therefore, this selected set of HLA-specific peptides allows vaccination against CRC for about 95% of Caucasian patients. So far, we detected specific T cell reactivity against two peptides (detection of 10-fold and 17-fold increase of IFNγ-producing T cells representing spots as compared to background in enzyme linked immunospot assays - ELISPOT) in CRC patients.Conclusions: We here provide for the first time a comprehensive evaluation of HLA-ligandomes in CRC across the most frequent Caucasian HLA-alleles. We developed a HLA-allotype specific warehouse of newly identified CRC associated HLA-ligands, which can be individually assembled according to patient specific HLA-alleles. This approach expands the applicability of peptide-based cancer vaccines to literally every CRC patient. DisclosuresNo relevant conflicts of interest to declare.

What makes children outgrow food allergy?

What makes children outgrow food allergy?

Natural killer cells facilitate PRAME-specific T-cell reactivity against neuroblastoma.

Neuroblastoma is the most common solid tumor in children with an estimated 5-year progression free survival of 20-40% in stage 4 disease. Neuroblastoma actively avoids recognition by natural killer (NK) cells and cytotoxic T lymphocytes (CTLs). Although immunotherapy has gained traction for neuroblastoma treatment, these immune escape mechanisms restrain clinical results. Therefore, we aimed to improve neuroblastoma immunogenicity to further the development of antigen-specific immunotherapy against neuroblastoma. We found that neuroblastoma cells significantly increase surface expression of MHC I upon exposure to active NK cells which thereby readily sensitize neuroblastoma cells for recognition by CTLs. We show that oncoprotein PRAME serves as an immunodominant antigen for neuroblastoma as NK-modulated neuroblastoma cells are recognized by PRAMESLLQHLIGL/A2-specific CTL clones. Furthermore, NK cells induce MHC I upregulation in neuroblastoma through contact-dependent secretion of IFNγ. Our results demonstrate remarkable plasticity in the peptide/MHC I surface expression of neuroblastoma cells, which is reversed when neuroblastoma cells experience innate immune attack by sensitized NK cells. These findings support the exploration of NK cells as adjuvant therapy to enforce neuroblastoma-specific CTL responses.

Abstract B33: Endoplasmic reticulum stress enhances the antigen-specific T cell immune responses and therapeutic antitumor effects generated by therapeutic HPV vaccines

Abstract Endoplasmic reticulum stress has a profound effect on cancer cell proliferation and survival, and also has the capacity to activate cells of the adaptive immune system. Multimodality treatments that combine conventional cancer therapies with antigen-specific immunotherapy have emerged as promising approaches for the control of cancer. However, it is not well known whether ER stress inducing agents can influence the efficacy of tumor antigen targeted vaccines. We used previously developed therapeutic human papillomavirus (HPV) DNA vaccines encoding calreticulin (CRT) linked to HPV-16 E7 antigen (CRT/E7) vaccine and ER stress inducing agent, 3-bromopyruvate in a preclinical model for its potential to enhance E7-specific CD8+ T cell immune responses as well as antitumor effects against E7-expressing tumor (TC-1 cells). In vitro assay, luciferase-expressing TC-1 tumor cells were treated with 3-bromopyruvate. E7-specific CD8+ T cells were added to evaluate the cytotoxicity. The degree of cytotoxic T lymphocytes-mediated killing of the tumor cells was measured with the IVIS Luminescence Imaging System. We also determined the levels of ER stress markers in 3-bromopyruvate treated TC-1 cells. In vivo assay, TC-1 tumor-bearing mice were treated with 3-bromopyruvate (10mg/kg, intraperitoneal injection) and/or CRT/E7 DNA vaccine (30μg/mouse). The tumor mass size and survival were evaluated. Treatment of TC-1 cells with 3-bromopyruvate induced significant in vitro cytotoxicity. We found that treatment with 3-bromopyruvate led to upregulation of ER stress markers (CHOP, GRP78) in E7-expressing TC-1 tumor cells. More importantly, treatment with combination of 3-bromopyruvate and CRT/E7 DNA vaccine led to improved antigen-specific CD8+ T cell immune responses as well as therapeutic antitumor effects in TC-1 tumor-bearing mice. Thus, our data indicate that 3-bromopyruvate can enhance therapeutic HPV vaccine potency in generating improved antigen-specific immune responses and antitumor effects. These findings have important implications for future clinical translation. Citation Format: Sung Yong Lee, JaeJeong Shim, Kang Kyung Ho, Kye Young Lee, Chien-Fu Hung, T.-C. Wu. Endoplasmic reticulum stress enhances the antigen-specific T cell immune responses and therapeutic antitumor effects generated by therapeutic HPV vaccines. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr B33.

Combination viroimmunotherapy with checkpoint inhibition to treat glioma, based on location-specific tumor profiling.

Systemic delivery of a complementary cDNA library expressed from the vesicular stomatitis virus (VSV) treats tumors by vaccinating against a wide range of tumor associated antigens (TAAs). For subcutaneous B16 melanomas, therapy was achieved using a specific combination of self-TAAs (neuroblastoma-Ras, cytochrome c, and tyrosinase-related protein 1) expressed from VSV. However, for intracranial B16 tumors, a different combination was therapeutic (consisting of VSV-expressed hypoxia-inducible factor [HIF]-2α, Sox-10, c-Myc, and tyrosinase-related protein 1). Therefore, we tested the hypothesis that tumors of different histological types growing in the brain share a common immunogenic signature which can be exploited for immunotherapy. Syngeneic tumors, including GL261 gliomas, in the brains of immune competent mice were analyzed for their antigenic profiles or were treated with systemic viroimmunotherapy. Several different histological types of tumors growing intracranially, as well as freshly resected human brain tumor explants, expressed a HIF-2α(Hi) phenotype imposed by brain-derived CD11b+ cells. This location-specific antigen expression was exploited therapeutically against intracranial GL261 gliomas using systemically delivered VSV expressing HIF-2α, Sox-10, and c-Myc. Viroimmunotherapy was enhanced by immune checkpoint inhibitors, associated with the de-repression of antitumor T-helper cell type 1 (Th1) interferon-γ and Th17 T cell responses. Since different tumor types growing in the same location in the brain share a location-specific phenotype, we suggest that antigen-specific immunotherapies should be based upon expression of both histological type-specific tumor antigens and location-specific antigens. Our findings support clinical application of VSV-TAA therapy with checkpoint inhibition for aggressive brain tumors and highlight the importance of the intracranial microenvironment in sculpting a location-specific profile of tumor antigen expression.

Topical steroid therapy induces pro-tolerogenic changes in Langerhans cells in human skin.

We have investigated the efficacy of conditioning skin Langerhans cells (LCs) with agents to promote tolerance and reduce inflammation, with the goal of improving the outcomes of antigen-specific immunotherapy. Topical treatments were assessed ex vivo, using excised human breast skin maintained in organ bath cultures, and in vivo in healthy volunteers by analysing skin biopsies and epidermal blister roof samples. Following topical treatment with a corticosteroid, tumour necrosis factor-α levels were reduced in skin biopsy studies and blister fluid samples. Blister fluid concentrations of monocyte chemoattractant protein-1, macrophage inflammatory proteins -1α and 1β and interferon-γ inducible protein-10 were also reduced, while preserving levels of interleukin-1α (IL-1α), IL-6, IL-8 and IL-10. Steroid pre-treatment of the skin reduced the ability of LCs to induce proliferation, while supernatants showed an increase in the IL-10/interferon-γ ratio. Phenotypic changes following topical steroid treatment were also observed, including reduced expression of CD83 and CD86 in blister-derived LCs, but preservation of the tolerogenic signalling molecules immunoglobulin-like transcript 3 and programmed death-1. Reduced expression of HLA-DR, CD80 and CD86 were also apparent in LCs derived from excised human skin. Topical therapy with a vitamin D analogue (calcipotriol) and steroid, calcipotriol alone or vitamin A elicited no significant changes in the parameters studied. These experiments suggest that pre-conditioning the skin with topical corticosteroid can modulate LCs by blunting their pro-inflammatory signals and potentially enhancing tolerance. We suggest that such modulation before antigen-specific immunotherapy might provide an inexpensive and safe adjunct to current approaches to treat autoimmune diseases.

Induction of Oral Tolerance with Transgenic Plants Expressing Antigens for Prevention/Treatment of Autoimmune, Allergic and Inflammatory Diseases.

The prevalence and incidence of autoimmune and allergic diseases have increased dramatically over the last several decades, especially in the developed world. The treatment of autoimmune and allergic diseases is typically with the use of non-specific immunosuppressive agents that compromise the integrity of the host immune system and therefore, increase the risk of infections. Antigenspecific immunotherapy by reinstating immunological tolerance towards self antigens without compromising immune functions is a much desired goal for the treatment of autoimmune and allergic diseases. Mucosal administration of antigen is a long-recognized method of inducing antigen-specific immune tolerance known as oral tolerance, which is viewed as having promising potential in the treatment of autoimmune and allergic diseases. Plant-based expression and delivery of recombinant antigens provide a promising new platform to induce oral tolerance, having considerable advantages including reduced cost and increased safety. Indeed, in recent years the use of tolerogenic plants for oral tolerance induction has attracted increasing attention, and considerable progress has been made. This review summarizes recent advances in using plants to deliver tolerogens for induction of oral tolerance in the treatment of autoimmune, allergic and inflammatory diseases.